Cephalosporin antibiotic compositions

ABSTRACT

Process and intermediate products useful in the preparation of cephalosporin compounds having a substituent at the 7-position in place of hydrogen are provided. The new cephalosporin compounds are active against various gram-negative and gram-positive organisms.

This is a division of application Ser. No. 149,364, filed June 2, 1971,now U.S. Pat. No. 4,297,488.

This invention relates to new antibiotics, new intermediate productsuseful in the preparation of these antibiotics, and processes for thepreparation of these compounds. More particularly, it is concerned withnew 7-aminocephalosporanic acid derivatives having a substituent atposition 7, and with new intermediates and processes for theirproduction.

The discovery of penicillin, which was found to be such an important andeffective antibiotic, stimulated great interest in this field.Subsequently, various other antibiotics such as streptomycin, thetetracyclines, novobiocin, and the like were found which greatlyincreased the doctors' armamentarium for treating infections due to avariety of pathogens. Unfortunately, the use of these antibiotics gaverise to strains of pathogens resistant to these known antibiotics. Inaddition, the known antibiotics suffer from the disadvantage that theyare only effective against certain types of microorganisms and are noteffective against a broad range of pathogens. Accordingly, the searchfor other antibiotics has continued.

It is an object of this invention to provide new cephalosporins havingantibiotic activity. A further object is to provide processes for thepreparation of these new antibiotics. Another object is to provide newintermediates useful in preparing these new cephalosporins. Otherobjects will be apparent from the detailed description of this inventionhereinafter provided.

The new cephalosporins of the present invention are compounds whereinthe Δ³ -cepham nucleus, namely a dehydrothiazine ring with a fusedβ-lactam, contains a substituent at the 7 position. Thus, these newcephalosporins which can be represented by the structural formula##STR1## wherein R' represents an acyl group, A represents an organicradical or group, and R₁ represents a radical or group replacinghydrogen, and the derivatives thereof such as esters, amides and saltsare valuable new antibiotic substances.

The acyl radical represented by R' can be a substituted or unsubstitutedaliphatic, aromatic or heterocyclic, araliphatic orheterocyclylaliphatic carboxylic acid radical or a carbothioic acidradical such as the acyl radicals of the known cephalosporins andpenicillins. These acyl radicals can be represented by the generalformula ##STR2## where R₂ is a radical of the group defined below, m andn represent 0-4 and R₃ represents R" or ZR", which are defined below.

One group of acyl radicals can be represented by the acyl group generalformula ##STR3## wherein R" represents a substituted or unsubstitutedstraight or branched chain alkyl, alkenyl or alkynyl group; aryl,aralkyl; cycloalkyl; or a heteroaryl or heteroaralkyl group. Thesegroups can be unsubstituted or can be substituted by radicals such asalkyl, alkoxy, halo, cyano, carboxy, sulfoamino, carbamoyl, sulfonyl,azido, amino, substituted amino, haloalkyl, carboxyalkyl,carbamoylalkyl, N-substituted carbamoylalkyl, guanidino, N-substitutedguanidino, guanidinoalkyl, and the like. Representative examples of suchacyl groups that might be mentioned are those wherein R" is benzyl,p-hydroxybenzyl, 4-amino-4-carboxybutyl, methyl, cyanomethyl,2-pentenyl, n-amyl, n-heptyl, ethyl, 3- or 4-nitrobenzyl, phenethyl,β,β-diphenylethyl, methyldiphenylmethyl, triphenylmethyl,2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl,3,5-dimethyl-4-isoxazolyl, 3-butyl-5-methyl-4-isoxazolyl,5-methyl-3-phenyl-4-isoxazolyl,3-(2-chlorophenyl)-5-methyl-4-isoxazolyl,3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl, D-4-amino-4-carboxybutyl,D-4-N-benzoylamino-4-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl,m-aminobenzyl, (3-pyridyl)methyl, 2-ethoxy-1-naphthyl,3-carboxy-2-quinoxalinyl,3-(2,6-dichlorophenyl)-5-(2-furyl)-4-isoxazolyl, 3-phenyl-4-isoxazolyl,5-methyl- 3-(4-quanidinophenyl)-4-isoxazolyl, 4-guanidinomethylphenyl,4-guanidinomethylbenzyl, 4-guanidinobenzyl, 4-guanidinophenyl,2,6-dimethoxy-4-guanidinophenyl, o-sulfobenzyl, p-carboxymethylbenzyl,p-carbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl,p-methoxybenzyl, 1-naphthylmethyl, 3-isothiazolylmethyl,4-isothiazolylmethyl, 5-isothiazolylmethyl, 4-pyridylmethyl,5-isoxazolylmethyl, 4-methoxy-5-isoxazolylmethyl,4-methyl-5-isoxazolylmethyl, 1-imidazolylmethyl, 2-benzofuranylmethyl,2-indolylmethyl, 2-phenylvinyl, 2-phenylethynyl,2-(5-nitrofuranyl)vinyl, phenyl, o-methoxyphenyl, o-chlorophenyl,o-phenylphenyl, p-aminomethylbenzyl, 1-(5-cyanotriazolyl)methyl,difluoromethyl, dichloromethyl, dibromomethyl,1-(3-methylimidazolyl)methyl, 2- or 3-(5-carboxymethylthienyl)methyl, 2-or 3-(4-carbamoylthienyl)methyl, 2- or 3-(5-methylthienyl)methyl, 2- or3-(5-methoxythienyl)methyl, 2- or 3-(4-chlorothienyl)methyl, 2- or3-(5-sulfothienyl)methyl, 2- or 3-(5-carboxythienyl)methyl,3-(1,2,5-thiadiazolyl)methyl, 3-(4-methoxy-1,2,5-thiadiazolyl)methyl,2-furylmethyl, 2-(5-nitrofuryl)methyl, 3-furylmethyl, 2-thienylmethyl,3-thienylmethyl, and tetrazolylmethyl.

The acyl group can also be a radical of the formula ##STR4## wherein nis 0-4, Z represents oxygen or sulfur, and R" is defined as above.Representative members of the substituent ##STR5## that might bementioned are allylthiomethyl, phenylthiomethyl, butylmercaptomethyl,α-chlorocrotylmercaptomethyl, phenoxymethyl, phenoxyethyl, phenoxybutyl,phenoxybenzyl, diphenoxymethyl, dimethylmethoxymethyl,dimethylbutoxymethyl, dimethylphenoxymethyl, 4-guanidinophenoxymethyl,4-pyridylthiomethyl, p-(carboxymethyl)phenoxymethyl,p-(carboxymethyl)phenylthiomethyl, 2-thiazolylthiomethyl,p-(sulfo)phenoxymethyl, p-(sulfo)phenylthiomethyl,p-(carboxy)phenoxymethyl, p-(carboxy)phenylthiomethyl,p-(carboxymethyl)phenoxymethyl, p-(carboxymethyl)phenylthiomethyl,2-pyrimidinylthiomethyl, phenethylthiomethyl,1-(5,6,7,8-tetrahydronaphthyl)oxomethyl, 6,8-bis(methylthio)octanoyl.

Alternatively, the acyl group can be a radical of the formula ##STR6##wherein R" is defined as above and R''' is a radical such as amino,hydroxy, azido, carbamoyl, guanidino, acyloxy, halo, sulfamino,tetrazolyl, sulfo, carboxy, carbalkoxy, and the like. Representativemembers of the substituent ##STR7## that might be mentioned areα-aminobenzyl, α-amino-2-thenyl, α-methylaminobenzyl,α-amino-γ-methylmercaptopropyl, α-amino-3 or 4-chlorobenzyl, α-amino-3or 4-hydroxybenzyl, α-amino-2,4-dichlorobenzyl,α-amino-3,4-dichlorobenzyl, D(-)-α-hydroxybenzyl, α-carboxybenzyl,α-amino-3-thenyl, α-amino-2-thenyl,D(-)-α-amino-3-chloro-4-hydroxybenzyl, D(-)-α-amino-3-thenyl,1-aminocyclohexyl, α-(5-tetrazolyl)benzyl, α-sulfaminobenzyl,α-sulfamino-3-thenyl, α-(N-methylsulfamino)benzyl,D(-)-α-guanidino-2-thenyl, D(-)-α-guanidinobenzyl, α-guanylureidobenzyl,α-hydroxybenzyl, α-azidobenzyl, α-fluorobenzyl,4-(5-methoxy-1,3-oxadiazole)-aminomethyl,4-(5-methoxy-1,3-oxadiazole)-hydroxymethyl,4-(5-methoxy-1,3-oxadiazole)-carboxymethyl,4-(5-methoxy-1,3-sulfadiazole)-aminomethyl,4-(5-methoxy-1,3-sulfadiazole)-hydroxymethyl,4-(5-methoxy-1,3-sulfadiazole)-carboxymethyl,2-(5-chlorothienyl)-aminomethyl, 2-(5-chlorothienyl)-hydroxymethyl,2-(5-chlorothienyl)-carboxymethyl, 3-(1,2-thiazole)-aminomethyl,3-(1,2-thiazole)-hydroxymethyl, 3-(1,2-thiazole)-carboxymethyl,2-(1,4-thiazolyl)-aminomethyl, 2-(1,4-thiazolyl)-hydroxymethyl,2-(1,4-thiazolyl)-carboxymethyl, 2-benzothienylaminomethyl,2-benzothienylhydroxymethyl, 2-benzothienylcarboxymethyl,2-azidooctyl-3-phenyl-3-azidomethyl, α-sulfobenzyl, andα-phosphonobenzyl.

Alternatively, the group ##STR8## can be a sulfonamido group such asphenylsulfonamido, ethylsulfonamido, benzylsulfonamido,2,5-dimethylsulfonamido, 4-chlorosulfonamido, 4-chlorophenylsulfonamido,4-methoxysulfonamido, and the like.

The acyl substituents of the general formula

    R.sub.11 R.sub.10 CHCO

wherein R₁₀ and R₁₁ are as defined below represent a preferred group ofsubstituents because of their generally useful antibiotic activity. R₁₀represents hydrogen, halo, amino, guanidino, phosphono, hydroxy,tetrazolyl, carboxy, sulfo or sulfamino. R₁₁ represents phenyl,substituted phenyl, a monocyclic heterocyclic 5- or 6-membered ringcontaining one or more oxygen, sulfur or nitrogen atoms in the ring,substituted heterocycles, phenylthio, heterocyclic or substitutedheterocyclic thio groups; or cyano. The substituents can be halo,carboxymethyl, guanidino, guanidinomethyl, carboxamidomethyl,aminomethyl, nitro, methoxy or methyl. Examples of these preferredsubstituents that might be mentioned are phenacetyl,3-bromophenylacetyl, p-aminomethylphenylacetyl,4-carboxylmethylphenylacetyl, 4-carboxamidomethylphenylacetyl,2-furylacetyl, 5-nitrofurylacetyl, 3-furylacetyl, 2-thienylacetyl,5-chlorothienylacetyl, 5-methoxythienylacetyl,α-guanidino-2-thienylacetyl, 3-thienylacetyl, 4-methylthienylacetyl,3-isothiazolylacetyl, 4-methoxyisothiazolylacetyl, 4-isothiazolylacetyl,3-methylisothiazolylacetyl, 5-isothiazolylacetyl,3-chloroisothiazolylacetyl, 3-methyl-1,2,5-oxadiazolylacetyl,1,2,5-thiadiazolyl-4-acetyl, 3-methyl-1,2,5-thiadiazolyl-4-acetyl,3-chloro-1,2,5-thiadiazolyl-4-acetyl,3-methoxy-1,2,5-thiadiazolyl-4-acetyl, phenylthioacetyl,4-pyridylthioacetyl, cyanoacetyl, tetrazolylacetyl,α-fluorophenylacetyl, D-phenylglycyl, 3-hydroxy-D-phenylglycyl,2-thienylglycyl, 3-thienylglycyl, phenylmalonyl, 3-chlorophenylmalonyl,2-thienylmalonyl, 3-thienylmalonyl, α-phosphonophenylacetyl,α-sulfaminophenylacetyl, α-hydroxyphenylacetyl, α-tetrazolylphenylacetyland α-sulfophenylacetyl.

The substituent A in formula I above can be hydrogen, hydroxy, halo,mercapto, acyloxy, acylthio, substituted hydroxy, substituted mercapto,a quaternary ammonium group, azido, amino or a N-substituted aminogroup. Alternatively, CH₂ A can be replaced by a formyl group.

Thus, CH₂ A can be a halomethyl such as chloromethyl, bromomethyl orfluoromethyl.

When A is a substituted hydroxy or substituted mercapto group, it can beshown by the formula

    --CH.sub.2 ZR.sub.5

where Z is oxygen or sulfur, and R₅ is an acyl group; a straight chainor branched chain loweralkyl, alkenyl or alkynyl group; an aryl group;an aralkyl group; or a heterocyclic group such as heteroaryl orheteroalkyl. These groups can be unsubstituted or can be substituted byradicals such as alkyl, alkoxy, halo, cyano, carboxy, carbamoyl, azido,sulfo, amino, substituted amino, haloalkyl, carboxyalkyl,carbamoylalkyl, N-substituted carbamoylalkyl, guanidino, N-substitutedguanidino, guanidoalkyl, sulfamyl, substituted sulfamyl, and the like.Representative of the groups thus represented that might be mentionedare methoxymethyl, n-propoxymethyl, methylthiomethyl, acetoxymethyl,propionyloxymethyl, benzoyloxymethyl, (p-chlorobenzoyl)oxymethyl,(p-methylbenzoyl)oxymethyl, pivaloyloxymethyl,(1-adamantyl)carboxymethyl, butanoyloxymethyl, carbamoyloxymethyl,(N-methylcarbamoyl)oxymethyl, (N-ethylcarbamoyl)oxymethyl,[N-(2-chloroethyl)carbamoyl]oxymethyl, (N-phenylcarbamoyl)oxymethyl,(N-p-sulfophenylcarbamoyl)oxymethyl,p-carboxymethylphenylcarbamoyloxymethyl, methoxycarbonyloxymethyl,isobutanoyloxymethyl, cyclobutylcarbonyloxymethyl, carbamoylthiomethyl,(ethoxythiocarbonyl)thiomethyl, (n-propoxythiocarbonyl)thiomethyl,(cyclopentanoxythiocarbonyl)thiomethyl, methylthiomethyl,N,N-diethylthiocarbamoylthiomethyl,N-methylpiperazinium-1-thiocarbonylthiomethyl,N,N-dimethylpiperazinium-1-thiocarbonylthiomethyl, 2-furoylthiomethyl,isothiouroniummethyl, (5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl,p-tolylsulfonylthiomethyl, mesyloxymethyl,1-methyl-1,2,3,4-tetrazolyl-5-thiomethyl, tosyloxymethyl,sulfamoyloxymethyl, 1-naphthoyloxymethyl, 2-furylacetoxymethyl,cinnamoyloxymethyl, p-hydroxycinnamoyloxymethyl,p-sulfocinnamoyloxymethyl and 1R:2S-epoxypropylphosphonyloxymethyl.

Alternatively, when CH₂ A is hydroxymethyl, the cephalosporin can alsoexist as the lactone which is formed by internal esterification with thecarboxy group.

The substituent CH₂ A can also be a group of the general formula

    --CH.sub.2 Y.sub.1

wherein Y₁ represents amino or substituted amino including nitrogenheterocycles and substituted heterocyclic groups. Examples of suchgroups that might be mentioned are aminomethyl, acetamidomethyl,carbamoylaminomethyl, N,N-dimethylaminomethyl,N-(2-chloroethyl)aminomethyl, 5-cyanotriazol-1-ylmethyl,4-methoxycarbonyltriazol-1-ylmethyl.

When A is amino the cephalosporin compound can also exist as the lactamformed by loss of water with the adjacent carboxy group.

Representative of the quaternary ammonium groups representing A thatmight be mentioned are pyridinium, 3-methylpyridinium,4-methylpyridinium, 3-chloropyridinium, 3-bromopyridinium,3-iodopyridinium, 4-carbamoylpyridinium,4-(N-hydroxymethylcarbamoyl)pyridinium,4-(N-carbomethoxycarbamoyl)pyridinium, 4-(N-cyanocarbamoyl)pyridinium,4-(carboxymethyl)pyridinium, 4-(hydroxymethyl)pyridinium,4-(trifluoromethyl)pyridinium, quinolinium, picolinium and lutidinium.

The preferred groups representing A are hydrogen, halo, azido, cyano,hydroxy, alkoxy, aryloxy, aralkyloxy, heterocycleoxy, mercapto,alkylthio, arylthio, aralkylthio, heterocyclethio, amino, alkylamino,alkanoylamino, hydroxyphenyl, acylthio, acyloxy, isothiouronium,sulfamoyloxy, quaternary ammonium, a heterocyclic tertiary amine,alkylsulfonyloxy and (cis-1,2-epoxypropyl)phosphono. The heterocyclescan be a 5 or 6 membered hetero ring containing one or more nitrogen,oxygen or sulfur atoms. The acyl group can be a loweralkanoyl group of2-6 carbon atoms, carbamoyl, or thiocarbamoyl and N-alkyl or N,N-dialkylderivatives thereof. The alkyl group of the foregoing substituentscontains 1-6 carbon atoms and may be further substituted radicals suchas alkoxy, halo, amino, cyano, carboxy, sulfo, and the like.

The substituent R₁ in formula I above can be hydroxy, mercapto orsubstituted hydroxy and mercapto groups; a hydrocarbyl or substitutedhydrocarbyl group; cyano, or a carbonyl or thiocarbonyl containingsubstituent bonded by said carbonyl or thiocarbonyl radical; a nitrogenbonded group; halo; or phosphono or a substituted phosphono group.

The oxy or thio substituent represented by R₁ in formula I can behydroxy or mercapto or a substituted hydroxy or mercapto group such as--XR'₁ wherein X is oxygen or sulfur and R'₁ is a hydrocarbyl group,preferably a straight or branched loweralkyl group of 1-6 carbon atoms,a straight or branched chain loweralkenyl or loweralkynyl group of 3-6carbon atoms, a monocyclic aryl group such as phenyl, or an aralkylgroup such as benzyl. These alkyl, alkenyl, alkynyl, aryl or aralkylgroups can be substituted with groups such as hydroxy, halo, nitro,amino, carboxy, sulfo, and the like. Other specific substituentsrepresented by R₁ that might be mentioned are groups of the formula--OCN, --SCN, --ONR₃ R₄, --SNR₃ R₄, --OAc, --SAc, --SO₃ H, --SO₃ R₂,--SO₂ NH₂, OCD₃, --SO₂ NR₃ R₄, --SO₂ R₂, --SO₂ NR₃ R₄, --OCOOR₂, --SOR₂,--OCOSR₂, --OCONR₃ R₄, and the like wherein Ac represents an acyl groupsuch as a loweralkanoyl, R₃ and R₄ represent hydrogen, loweralkyl, acyland loweralkoxy, and R₂ represents loweralkyl, haloloweralkyl, aryl,aralkyl and substituted derivatives of such groups.

When R₁ is hydrocarbyl or substituted hydrocarbyl it can be loweralkyl,loweralkenyl, loweralkynyl, aralkyl, cycloalkyl, a monocyclic arylgroup, or a monocyclic heterocyclic group which can also be substitutedwith one or more groups such as halo, hydroxy, alkoxy, amino, nitro,sulfonyl, sulfamoyl, acyloxy, carbamoyloxy, carboxy, carboxamido andN-substituted carboxamido.

R₁ in formula I above represents cyano or a group of the general formula--CX'R" wherein X' is oxygen or sulfur, and R" is hydrogen, halo,hydroxy, mercapto, amino, substituted amino, an aliphatic radical, anaromatic radical, an aliphatic-oxy radical or an aromatic-oxy radical.Examples of these substituents that might be mentioned are --COOH,--CSSH, --COR₂, --COOR₂, --COSR₂, --CSSR₂, --CONH₂, --CSNH₂, --CSR₂,--CONHR₂, --CSNH, --CONR₃ R₄ and --CSNR₃ R₄ wherein R₂ represents astraight or branched chain alkyl group of 1-6 carbon atoms and R₃ and R₄represent hydrogen or R₂.

R₁ in formula I above represents a nitrogen bonded group such as aminoand substituted amino groups, nitro, azido, nitroso, isocyanoto,isothiocyanato and hydroxyamino. Specific examples of nitrogen bondedgroups that might be mentioned are --NH₂, --NHR₂, --NHC(O)_(n) R₂,--NHC(S)_(n) R₂, --NR₂ R₃, --NHNH₂, --NHNR₂ R₃, ##STR9## --NNR₂, --NR₃OH, --NHCNHNH₂, --NHCNHNR₂ R₃, --NO₂, --NO, --NCO, N₃ and --NCS, whereinR₂ represents a straight or branched chain loweralkyl group of 1 to 6carbon atoms, R₃ represents R₂ or hydrogen, and n represents the integer1 or 2.

The substituent R₁ in formula I represents phosphono or a metal or aminesalt thereof, or a substituted phosphono group of the formula ##STR10##where Y' and Z' are the same or different and represent --OR₂, --NR₃ R₄,##STR11## --NC═X', --OCOR₂ and --N₃, where R₂ represents hydrogen or ahydrocarbyl radical, R₃ and R₄ represent hydrogen, hydrocarbyl, alkoxyor an acyl radical, and X' represents oxygen or sulfur.

In accordance with the nomenclature of cephalosporin compounds used inthe art, the compound obtained by hydrolysis of cephalosporin C, whichcan be represented by the structural formula ##STR12## is called7-aminocephalosporanic acid or 7-ACA.

The term "decephalosporanic acid" used herein to described certainproducts, pursuant to its usage in this art, represents the basicheterocyclic nucleus having the structural formula ##STR13##

Thus, a compound of the formula ##STR14## is called3-methyl-7-aminodecephalosporanic acid using this system ofnomenclature.

The cephalosporin compounds with which this invention is concerned arealso conveniently designated as "cepham" compounds containing the basicfused-ring betalactam thiazine structure ##STR15## which is known ascepham. Thus, the cephalosporin compounds are called "cephem" referringto the basic structure with a single olefin band. For example, in thissystem of nomenclature cephalosporin C having the structural formula##STR16## is named7-(5'-aminoadipamido)-3-acetoxymethyl-3-cepham-4-carboxylic acid.

In accordance with the present invention, it is now found that the newcephalosporins of this invention can be prepared by processes which canbe depicted as follows ##STR17## where R', R₁ and A are as definedabove.

In the foregoing flowsheet the starting compound is a derivative of7-aminocephalosporanic acid (II), hereinafter also called 7-ACA, whereinthe carboxy group is preferably blocked, for example by forming asuitable ester. Thus, 7-ACA or analogs thereof having a differentsubstituent at 3 can be esterified in accordance with methods well knownin this art to obtain, for example, the esters wherein R₈ represents analkyl or substituted alkyl group such as methyl, t-butyl,pivaloyloxymethyl, acetoxymethyl and the like, a haloalkyl such astrichloroethyl, an alkenyl group such as allyl, an alkynyl group such aspropargyl, an aralkyl group such as benzyl, benzhydryl, o-nitrobenzyl,3,5-dinitrobenzyl or p-methoxybenzyl, an aryl group such as phenacyl, anorganicmetallic group for example a silyl group such as trimethylsily,or a stannyl group such as tributyltin, phenacyl ortrichloroethoxycarbonyl. The ester (II) is converted to thecorresponding 7-diazocephalosporanic acid ester or 3-CH₂A-7-diazocephalosporanic acid ester (III) by reaction with nitrite. The7-diazo ester (III) is converted by reaction with a pseudo halogencompound or compounds, or a compound which acts as a pseudo halogen, toform intermediate product (IV) wherein X represents halogen from thegroup consisting of bromine, chlorine and iodine or another leavinggroup, and Y is a nitrogenous substituent or R₁. Intermediate compound(IV) is then converted to compound (V) wherein R₁ represents asubstituent other than hydrogen and Z represents a nitrogenous groupwhich is readily convertible to amino or acylamino. Compound (V) is thenconverted to the desired cephalosporin ester (VI) which can be reactedto obtain the corresponding cephalosporin acid or a salt thereof. Also,the substituent at position 3 of the Δ³ -cepham nucleus can be convertedto the other substituents of the formula --CH₂ A in accordance withmethods known in this art and those described herein. The processes forcarrying out the various steps of the foregoing flowsheet will be morereadily understood from the detailed descriptions of methods which canbe used to carry out these processes which follows.

The starting material in the foregoing process can be 7-ACA or a 3-CH₂A-7-aminodecephalosporanic acid which is first reacted to block orprotect the carboxy group. Examples of particularly suitable 3-CH₂A-7-aminodecephalosporanic acids that might be mentioned are thosewherein A represents hydrogen, hydroxy, azido, halo, a tertiary amine,isothiouronium, a loweralkoxy or loweralkylthio group, an acyloxy oracylthio group, or a heterocyclic oxy or heterocyclic thio substituent.When A is halo it can be fluoro, chloro or bromo. When A represents aloweralkoxy or loweralkylthio group it may be a group such as methoxy,methylthio, tertiary butyloxy, tertiary butylthio, and the like. When Arepresents an acyloxy or acylthio group it may be a group such asacetoxy, benzoyloxy, cinnamoyloxy, p-sulfocinnamoyloxy, isobutyryloxy,pivaloyloxy, adamantoyloxy, carbamoyloxy, n-methylcarbamoyloxy,N-p-sulfophenylcarbamoyloxy, N-p-carboxymethylphenylcarbamoyloxy,N-chloroethylcarbamoyloxy, N,N-diethyldithiocarbamoyloxy,N,N-dimethylpiperidinodithiocarbamoyloxy, mesyloxy, sulfamoyloxy and1R:2S-1,2-epoxypropylphosphonyloxy. When A is a heterocyclic oxy orheterocyclic thio group it may be a group such as5-methyl-1,3,4-thiadiazolyl-2-thio and4-carboxamido-1,3,4-thiadiazolyl-2-thio. When A represents a tertiaryamine it can be pyridinium and the like.

The diazotization of the 7-amino ester is carried out in accordance withprocesses well known in this art. Thus, it is conveniently effected inaqueous or aqueous-organic solvent medium, for example by reaction withsodium nitrite in the presence of acid or by reaction with an organicnitrite. Organic solvents suitable for carrying out this reaction arethose which do not contain an active hydrogen. Examples of such solventsthat might be mentioned are methylene chloride, ether, benzene, toluene,chloroform, and the like. The reaction is preferably carried out attemperatures between about 0° and 50° C.; usually it is mostconveniently effected at room temperature. The isolation of the desireddiazo compound is readily accomplished in accordance with methods knownin the art.

Thus, in accordance with one specific embodiment of this invention, thenew cephalosporins are obtained by the following processes: ##STR18##where the substituents are as defined above.

In the above process the 7-diazocephalosporanic acid ester (III) isreacted with a halo azide from the group consisting of bromine, chlorineor iodine azide, preferably in the presence of a tertiary amine azide,to produce the intermediate 7-halo-7-azidocephalosporanic acid ester(VII) which on reaction with a suitable nucleophilic reagent isconverted to the desired 7-R₁ -7-azidocephalosporanic acid ester (VIII).This intermediate product is reduced and acylated in one step to formthe substituted cephalosporanic ester (XI) which can then be cleaved toremove the blocking group and obtain the cephalosporanic acid or a saltthereof (X). Alternatively, as shown in the flowsheet, the 7-R₁-7-azidocephalosporanic acid ester (VIII) is reduced to the 7-R₁-7-aminocephalosporanic acid ester (IX) which can be acylated to producethe 7-R₁ -7-acylaminocephalosporanic acid ester (XI), or the ester groupof compound (IX) can be cleaved to obtain the free acid (X) which can beacylated to form the desired substituted cephalosporin or a saltthereof. The step of cleaving the blocking group is readily effected inaccordance with methods known in this art. For example, an aralkyl groupsuch as the benzyl ester is removed by reduction, a silyl ester can beremoved by hydrolysis to form the free acid or a salt thereof and abenzhydryl group is readily cleaved by reaction with trifluoroaceticacid in the presence of anisole. In this process other esters which arereadily cleaved to form the free acid such as trichloroethyl,phthalimidomethyl, succinimidomethyl, p-methoxybenzyl, o-nitrobenzyl,phenacyl and t-butyl and the like can be used. Also, as is discussedabove, the 3-substituent on the Δ³ -cepham nucleus can be variedfollowing the procedures known in this art to obtain the substitutedcephalosporins of formula I.

The step of producing the halo azide intermediate is carried out byreacting the diazo compound with a halo azide at a temperature betweenabout 0° and 50° C. for sufficient time to complete the formation of thedesired compound. The reaction is preferably carried out in a suitableorganic solvent medium which is inert to the reactants. Various solventswhich do not contain an active hydrogen such as methylene chloride,chloroform, benzene, toluene, ether and the like, or mixtures thereofprovide suitable mediums for carrying out the reaction. Generally, it ispreferred to effect the reaction in the presence of a second azide suchas lithium azide or a tertiary ammonium azide, for exampletriethylammonium azide, since under these conditions the formation ofthe undesired 7-dibromo compound is avoided. The halo azide is used inan amount in slight excess of stoichemetric requirements. The amount ofsecond azide is not critical and it is generally desirable to use anexcess in order to obtain maximum yields of the desired halo azidocompound under optimum conditions. After completion of the formation ofthe halo azide the product is recovered and can be purified further, forexample by chromatography, in accordance with processes well known inthis art.

The next step of the process comprising the replacement of the halosubstituent by a nucleophilic group is effected by reacting the haloazide with a substance capable of furnishing a group to replace thehalo. This reaction is preferably carried out in the presence of asuitable non-reactant solvent such as methylene chloride, chloroform,benzene, toluene, ether, petroleum ether and the like; again it isdesirable to avoid using any solvents containing an active hydrogen.Thus, in accordance with a specific embodiment of this invention, thenucleophilic displacement reagent can be an alcohol such as methanol,ethanol, phenol, benzyl alcohol, a substituted alcohol such as2-bromoethanol, 2-methoxyethanol, glycol amide, an ester of glycolicacid and the like which results in the displacement of the halo groupand the introduction of a methoxy, ethoxy, phenoxy, benzyloxy,2-bromoethoxy, methoxy, 2-methoxyethoxy, carbonylmethoxy or esterifiedcarbonylmethoxy substituent, respectively. The reaction is preferablycarried out in the presence of a heavy metal cation such as a silversalt.

When the reaction is carried out by reacting a salt of an organic acid,preferably a heavy metal salt such as a silver salt, the corresponding7-acyloxy compound is obtained. For example, reaction of the halo azidewith silver acetate, silver benzoate, silver t-butylacetate, silverphenylacetate the corresponding 7-acetoxy, 7-benzoyloxy,7-t-butylacetoxy and the 7-phenylacetoxy intermediate compound isobtained. The acyl groups of these various acyloxy compounds can then becleaved to obtain the corresponding 7-hydroxy compound. Alternatively,in this process of preparing the 7-acyloxy compounds the reaction can becarried out by using a salt of the appropriate acid and carrying out thereaction in the presence of a heavy metal salt such as silver oxide orsilver tetrafluoroborate.

In the next step of the above-described process the 7-azido-7-R₁compound is then reduced to afford the corresponding 7-amino-7-R₁compound. Various methods of carrying out this reduction can beemployed, but it is generally preferred to carry out the reduction ofthe azido to the amino group by catalytic hydrogenation employing anoble metal catalyst such as platinum, palladium or oxides thereof.These processes are carried out in accordance with procedures well knownin this art. Alternatively, the reduction can be effected in thepresence of a suitable acylating agent to produce the desired7-acylamino-7-R₁ compound. The 7-amino compound can be reacted withsuitable acylating agents using procedures well known in this art toobtain the desired 7-acylamido compounds. Thus, in the above-describedprocess where the substituent R is a halo group, for example chlorine,bromine or iodine, the 7-azido-7-halo compound can be reduced to thecorresponding amine compound and the latter compound can then beacylated to obtain the 7-acylamino-7-halo product. Alternatively, asdiscussed above, the reduction and acylation steps can be combined toproduce the 7-acylamido compound without separating and acylating the7-acylamido intermediate.

Those 7-amidocephalosporanate products wherein the substituent inposition 7 of the cepham nucleus is bonded to the 7-carbon via anitrogen atom are conveniently synthesized from their corresponding7-halo-7-azido precursors. According to this method of preparation a7-halo-7-azidocephalosporanate is converted to the corresponding7,7-diazidocephalosporanate via treatment with an alkali metal azide andthis intermediate is then subjected to reduction via hydrogenation inthe presence of a suitable catalyst as, for example, apalladium-on-charcoal catalyst. The resulting7-amino-7-azidocephalosporanate is then acylated by treatment with anacyl halide, carboxylic acid anhydride or sulfonyl halide and the7-amido-7-azidocephalosporanate thus obtained is again subjected toreduction and then converted to the free acid by conventional means toafford the desired product. The following equation, wherein theacylating agent employed is an acyl halide, illustrates this method ofpreparation; however, it is to be understood that any other acylatingagent can be substituted therefor in an otherwise analogous reaction toafford the desired 7-amido- or 7-sulfonamidocephalosporanic acidproduct: ##STR19##

The 7-amido-7-aminocephalosporanic acids of this invention areintermediates which will react at the amino nitrogen atom with a widevariety of reagents to afford the N-substituted and N,N-disubstitutedderivatives thereof. Thus, for example, a 7-amido-7-aminocephalosporanicacid will react with one or more equivalents of an aldehyde such asformaldehyde, acetaldehyde or propionaldehyde and the like or anaralkaldehyde such as benzaldehyde and the like to afford thecorresponding 7-amido-7-N-alkyl(or aralkyl)cephalosporanic acid.

In addition to the reaction with aldehydes a7-amido-7-aminocephalosporanic acid can be treated with an acylating andsulfonating agent such as an acyl halide, carboxylic acid anhydride,alkanesulfonyl halide or pyridine-sulfur trioxide complex to afford thecorresponding 7-amido-7-acylamido(or 7-sulfonamido)cephalosporanic acidproduct.

Those 7-amido-7-aminocephalosporanic acids wherein the 7-amino radicalis substituted by ureido or an N,N-dialkylureido are convenientlyobtained by treating the former with the appropriate carbamoyl halide orN,N-dialkylcarbamoyl halide. Similarly, the7-amido-7-guanidinocephalosporanic acid derivatives are obtained bysimply treating the 7-amido-7-aminocephalosporanic acid precursor withN-amidino-3,5-dimethylpyrazole.

The 7-amido(7-amidinoureido)cephalosporanic acid derivatives areobtained by first treating the 7-amido-7-aminocephalosporanic acidprecursor with phosgene to afford an intermediate which, upon treatmentwith guanidine, yields the desired product.

Alternatively, in accordance with a further embodiment of this inventionthe 7-aminocephalosporins are also obtained using a benzhydryl ester ofthe 7-azido-7-halo compound of formula VII as the starting material.This compound is reacted with t-butyl carbamate to produce thecorresponding 7-t-butylcarbonylamino compound. Reduction of thisintermediate product with hydrogen in the presence of platinum oxideaffords the 7-amino-7-t-butylcarbamoylaminobenzhydryl ester. The lattercompound is then acylated to produce thebenzhydryl-7-acylamido-7-t-butylcarbamoylamino compound which ontreatment with trifluoroacetic acid in the presence of anisole affordsthe 7-aminocephalosporin.

The 7-amido-7-phosphono compounds and the 7-amido-7-phosphinyl productsof this invention and their corresponding salt and ester derivatives areobtained by treating a 7-azido-7-halocephalosporanate compound with anappropriate phosphite, phosphonamidic acid or diamidophosphorous acid inthe presence of a metal salt, i.e. a silver salt such as silver oxide orsilver tetrafluoroborate and the like. The 7-azido-7-phosphono (or7-phosphinyl) compound thus obtained is then reduced to thecorresponding 7-amino-7-phosphono (or 7-phosphinyl)cephalosporanate andsubjected to acylation via treatment with an acyl halide, carboxylicacid anhydride or sulfinyl halide to afford the corresponding7-amido-7-phosphono (or phosphinyl) compound and this intermediate canthen be isolated and purified or, if desired, the said ester may beconverted to the corresponding free acid as described above. Also, upontreatment with a base, the said acid can be converted to itscorresponding 7-amido-7-phosphono (or phosphinyl) salt.

When the halo azide compound is reacted with carbon dioxide or carbondisulfide in the presence of phenyllithium, the corresponding7-azido-7-carboxy or 7-azido-7-thiocarboxy compound is obtained. Thesecarboxy or thiocarboxy compounds can be converted to the correspondinghaloformyl compound by reaction with halogenating agents pursuant toprocesses well known in this art. For example, the 7-carboxy-7-azidocompound by reaction with thionyl chloride is converted to the7-chloroformyl-7-azido compound which can be reduced to the7-amino-7-chloroformyl compound and acylated to produce the desiredcephalosporanic acid or decephalosporanic acid compounds. Further, the7-haloformyl compound on reaction with an alcohol such as methanol,phenol or benzyl alcohol is converted to the corresponding7-methoxycarbonyl, 7-phenoxycarbonyl, or 7-benzyloxycarbonyl compound.Upon reacting the 7-haloformyl compound with an amine such asdimethylamine, dibenzylamine, diphenylamine, monoethylamine,monobenzylamine, monophenylamine, phenethylamine, hydrazine or asubstituted hydrazine is converted to the corresponding 7-carboxamidocompound. The 7-carboxycephalosporanic and decephalosporanic acidcompounds are also obtained by oxidizing the corresponding 7-formylcompounds with argentic oxide. The 7-formyl compounds are prepared bytreating the 7-hydroxymethyl substituted products with phosphoric acidat pH 2-3 to obtain the 7-hydroxy compound and then oxidizing theselatter products with chromium trioxide pyridine complex.

The new cephalosporanic and decephalosporanic acids wherein R₁ is ahydrocarbyl group are prepared by reactions shown in the followingflowsheet: ##STR20## where D is a hydrocarbyl and R₁ and A are asdefined above.

In accordance with the foregoing flowsheet, the diazocephalosporincompound is reacted with a trihydrocarbyl boron compound at lowtemperatures, i.e. -50° C. to -100° C., for sufficient time to producethe 7-dihydrocarbylboron-7-hydrocarbon intermediate (XVII). Uponreacting this intermediate with a halogen azide such as bromine azide atroom temperature, the 7-hydrocarbyl-7-azido compound (XVIII) isobtained. The latter compound is then reduced catalytically, acylatedand the ester group is cleaved in accordance with the proceduresdescribed above to produce the desired7-hydrocarbyl-7-acylamidocephalosporanic or decephalosporanic acid (XIX)or a salt thereof.

In carrying out the first step of this procedure, the hydrocarbyl groupof the boron compound can be a lower-alkyl group of 1 to 6 carbon atoms,a loweralkenyl group of 2 to 6 carbon atoms, a loweralkynyl group of 2to 6 carbon atoms, an aralkyl group such as benzyl, or an aryl groupsuch as phenyl. Thus, using these tri-substituted boron compounds, thecorresponding 7-alkyl, alkenyl, alkynyl, aralkyl or arylcephalosporanicacid compounds are obtained.

Thus, in accordance with a specific embodiment of this invention, newcephalosporins having a 7-carboxy or substituted carboxy substituent areobtained by the following processes: ##STR21##

Thus, pursuant to one of the foregoing processes, the intermediateproduct (IV) obtained as described above is reacted with a hydrocarbyllithium compound of the formula R₁₀ Li where R₁₀ represents ahydrocarbyl group such as loweralkyl or aryl, for example n-butyllithium, to form the 7-lithium compound (XX) which is reacted withcarbon dioxide to produce the 7-α-carboxy compound (XXI). Thisintermediate is converted to the carboxy 7-cephalosporin (XXII) usingmethods shown above, or the carboxy substituent can be converted to acarboxylic acid derivative such as an ester, an amide, a hydrazide, anazide or a hydroxamic acid using procedures known in the art.Alternatively, when the 7-lithium compound is reacted with carbondisulfide in place of carbon dioxide, the corresponding 7-dithiocarboxy(--CSSH) compound is obtained.

The 7-cyanocephalosporins are prepared by reacting the 7-halo-7-azidointermediate of formula VII above with tetrabutylammonium cyanide toobtain the 7-cyano-7-azido compound. This intermediate product is thenreduced to the 7-cyano-7-amino compound, the latter product is acylated,and the acylated ester is cleaved to obtain the desired7-cyano-7-acylamido cephalosporin using the procedures described above.

The 7-formyl cephalosporins are prepared by converting a7-hydroxymethyl-7-acylamido-cephalosporanic acid or a corresponding3-CH₂ A decephalosporanic acid with an oxidizing agent such aspyridine-chromium trioxide to produce the 7-formyl compound. This lattercephalosporin compound is converted to the corresponding 7-carboxyproduct by mild oxidizing agents such as argentic oxide.

The 7-halo-substituted cephalosporins of this invention are prepared bysubjecting the 7-halo-7-azido intermediates of formula VII above toreduction to afford the corresponding 7-halo-7-amino compound and thisintermediate is acylated to afford the corresponding7-acylamido-7-halocephalosporin compound. The resulting ester is thencleaved and converted to its corresponding carboxylate salt byconventional means as, for example, by treatment with trifluoroaceticacid and an aqueous solution of a base.

In another embodiment of this invention, the novel 7-hydrocarbyloxy and7-hydrocarbylthiocephalosporins can be obtained by the followingsequence: ##STR22## where R₈ and A are the same as defined above and Grepresents hydrocarbyloxy or hydrocarbylthio.

In accordance with the above flowsheet, the starting compound, an esterof a 7-diazo compound defined as in III above, is reacted with ahypohalite of an alcohol or a thiol, or with an alcohol in the presenceof a positive halogen such as a N-halo-amide, for example,N-bromoacetamide, N-bromosuccinimide, N-bromophthalimide and the like,that react as though they were the corresponding hypohalite. Theresultant 7-halo-7-hydrocarbyloxy or hydrocarbylthio ester (XXIII) isfrequently a mixture of epimers at 7, which are readily separable bychromatography. However, when only one epimer is obtained, it may beequilabrated to a mixture of epimers by treatment with an inorganichalide in a polar solvent. A lithium salt of the appropriate halide indimethylformamide is particularly useful for epimerizing theseintermediates. The 7-halo-7-hydrocarbyloxy or hydrocarbylthio productcan then be reacted with an azide, such as lithium azide, to form the7-hydrocarbyloxy or hydrocarbylthio-7-azidocephalosporanate ester(XXIV). This latter compound can then be reduced either with hydrogen oran inorganic reducing agent to form the intermediate 7-hydrocarbyloxy orhydrocarbylthio-7-amino ester (XXV) (R'═H). This latter compound can beacylated to produce the substituted cephalosporin ester. Alternatively,the reduction of the azido intermediate can be done in the presence ofan acylating agent to produce these esters directly. These compounds canthen be converted to the desired cephalosporin of formula XXV or saltsthereof in accordance with procedures described above.

The various processes described above can result in the production of aparticular epimer at 7, or in a mixture of epimers at 7; i.e., a7α-halo-7β-R₁ or a 7β-halo-7α-R₁ compound. When a mixture of epimers isobtained, these can be readily separated in accordance with methods,such as chromatography, which are well known in this art. In some cases,when only one epimer is obtained it can be equilabrated to produce amixture of epimers by procedures known in the art.

Pursuant to a further embodiment of this invention, novel products arealso obtained by a new process whereby the acyl group of a7-acylamidocephalosporin compound is replaced by a different acylsubstituent. In accordance with this new process, the7-acylamidocephalosprin compound is reacted with an acylating agent toobtain an intermediate 7-diacylamidocephalosporin compound containingtwo different acyl substituents, and the original acyl group is thencleaved to obtain a new 7-acylamidocephalosporin compound. This processis illustrated in the following flowsheet: ##STR23## wherein Acrepresents an acyl group, A', R'₁ and R_(p) represent, respectively,substituents defined as A, R₁ and R', respectively, or are reconvertiblethereto by the removal of any protecting or blocking group.

In the process described in the foregoing flowsheet, the reactions canbe carried out with the free acid, although in general it is foundpreferable to block or protect the carboxy group by the formation of asuitable ester which can be readily removed at the end of the process.

The first step of this process comprises reacting the cephalosporincompound, or a derivative thereof wherein the carboxyl group is blocked,with an acylating agent, preferably an acyl halide, in the presence of asilyl group to produce the 7-diacylamido compound. This product is thenreacted to remove the original acyl substituent and produce thecephalosporin compound having the new 7-acylamido substituent.

The first step of producing the diacylated product is best effected byintimately contacting the cephalosporin compound with an acylating agentin a suitable solvent medium in the presence of a tri-substituted silylderivative of a negatively-substituted amide. The temperature at whichthe reaction is carried out is not particularly critical andtemperatures from about -20° C. to about 100° C. are generallysatisfactory, although we prefer to carry out the reaction attemperatures from about 25° to 40° C. Various solvents which do notcontain an active hydrogen such as chloroform, acetonitrile, methylenechloride, dioxane, benzene, halobenzene, carbon tetrachloride, anddiethylether are most suitable as mediums in the reaction.

Various trihydrocarbylsilyl compounds in which the hydrocarbylsubstituent is a loweralkyl (1 to 6 carbon atoms), an aryl such asphenyl, or an aralkyl group such as benzyl can be utilized in theprocess of this invention. These compounds are readily prepared byreacting equimolecular amounts of a trihydrocarbylsilyl halide with anegatively-substituted amide or imide. However, it is generallypreferred to use a triloweralkylsilyl derivative, and in particular thetrimethylsilyl derivative since this product is inexpensive and readilyavailable. Negatively-substituted amides and imides that might bementioned are succinamide, phthalimide, cyanoacetamide,trifluoroacetamide, benzamide, p-nitrobenzamide, trichloroacetamide, asulfonamide, and the like. Examples of triloweralkylsilyl derivativesthat are especially useful and might be mentioned areN-trimethylsilyltrifluoroacetamide, N-trimethylsilylphthalamide.

Generally, it is preferred to carry out the foregoing reactions with acephalosporin compound wherein the carboxy group is blocked or protectedsince maximum yields of the desired product are obtained with suchderivatives. For this purpose, the carboxy substituent is blocked byforming a suitable ester such as a benzyl, benzhydryl, p-nitrophenyl,trimethylsilyl, trichloroethoxy, p-methoxybenzyl, phthalimidomethyl, orsuccinimidomethyl ester which are readily removed by processes wellknown in this art. In addition, it is generally preferred to block orprotect any amino groups present in the starting cephalosporin compoundsince maximum yields of the desired products are obtained with suchderivatives. For this purpose, the groups are preferably blocked withsubstituents that are readily removed. Such groups are well known in theart. For example, the amino group is most conveniently blocked by agroup such as trichloroethoxycarbonyl, t-butoxycarbonyl,benzoylmethoxycarbonyl, trimethylsilyl, p-methoxybenzyloxy,o-nitrophenylthio, and the like.

The step of cleaving the original acyl group can be effected in severalways, namely, by prolonging the reaction time, by the addition of analcohol such as a loweralkanol or a loweralkylthiol, or by hydrolysis inan aqueous solution containing a small amount of an acid or a base.Thus, in some cases cleavage is effected by the addition of aloweralkanol or loweralkylthiol containing from 1 to 6 carbon atoms, anaralkanol such as benzyl alcohol, or the corresponding thiol. Thecleavage affords the desired monoacylated cephalosporin compound or canalso result in the production of a mixture of the monacylated compounds.In the latter case the desired monoacylated cephalosporin compound isrecovered by separation procedures such as chromatography which are wellknown in this art.

The process of this embodiment of our invention is particularly suitablefor replacing the aminoadipoyl group of 7-(aminoadipoylamido) side chainof cephalosporins such as those obtained by fermentation and derivativesthereof having other substituents at the 3 position. Thus, in accordancewith a specific embodiment of this process, a cephalosporin compoundsuch as cephalosprin C or7-(D-5'-amino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid or derivatives thereof, is reacted with an acylating agent in thepresence of a tri-substituted silyl radical to obtain the 7-diacylamidoderivative having two different acyl groups. The diacylated product canbe selectively cleaved to remove the α-aminoadipoyl group and obtain thedesired different 7-acylamidocephalosporin compound. Although thecephalosporin compound per se can be transesterified by our process, wehave found that the process is facilitated and maximum yields of the new7-acylamido compound are obtained under optimum conditions when theamino and the carboxy substituents of the cephalosprin compound areblocked or protected in carrying out our process. The various blockingor protecting groups mentioned above are suitable for this purpose.Thus, for example, in replacing the α-aminoadipoyl side chain of thecephalosporins mentioned above with another acyl group, pursuant to thispreferred embodiment of this invention, both the carboxy group at the4-position and the carboxy group of the aminoadipoyl substituent areblocked and the amino group is similarly protected. The resultingblocked derivative is reacted with an acylating agent, preferably anacid halide such as the chloride, in the presence of the tri-substitutedsilyl derivative of the negatively-substituted amide or imide to producethe 7-diacylamido derivative. During this acylation reaction somecleavage of the α-aminoadipoyl group occurs, but most of the product isobtained in the form of the diacylated derivative.

When the protecting group of the amino substituent of the aminoadipoylmoiety such as a trichloroethoxycarbonyl or a t-butoxycarbonyl group, isremoved by suitable means, a selective cleavage of the aminoadipoylgroup occurs. This removal of the protecting group of the amino functionapparently results in an internal cyclization of the aminoadipoyl groupresulting in cleavage of the grup as the α-carboxylic ester of theformula ##STR24## Our present evidence indicates that this is themechanism of this cleavage, however we do not wish to be bound by thisexplanation of how the cleavage occurs since subsequent studies mayestablish that the product is cleaved and extruded in some other manner.This explanation of how the cleavage occurs is presented to provide abetter understanding of our invention.

The cleavage of the protective groups on the amino and carboxy functionsis accomplished in accordance with procedures well known in this art.Thus, for example, the trichloroethoxy carbonyl group is removed byreaction with zinc and acetic acid, and the t-butoxycarbonyl andbenzhydryl groups are removed by reaction with trifluoroacetic acid.

In accordance with a further aspect of our invention, new 7-diacylamidocompounds obtained by our process are not only useful as intermediatesin the preparation of monoacylated cephalosporins but are usefulantimicrobial products active against various pathogenic microorganisms.

A more complete understanding of the processes of this invention isprovided by illustrative embodiments which follow. Thus,7-(D-5'-amino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid is converted to the corresponding 7-(2-thienylacetamido) compoundin accordance with the processes of the following sequence of reactions:

    ______________________________________                                         ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                

In the foregoing process the starting compound is acylated by reactionwith trichloroethoxycarbonyl chloride to produce theN-trichloroethoxycarbonyl derivative which, upon alkylation withdiphenyldiazomethane, is converted to the dibenzhydryl ester. Reactionof the resulting cephalosporin compound withtrimethylsilyltrifluoroacetamide and 2-thienylacetyl chloride affordsthe7-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-(2-thienylacetaylamino)]compound. This aminoadipoyl group is then cleaved by reaction with zincin the presence of acid to obtain the benzhydryl ester of3-carbamoyloxymethyl-7-(2-thienylacetamidodecephalosporanic acid, whichis further deblocked to remove the benzhydryl group and form the freeacid. This product can be converted to a salt in accordance with methodsknown in this art.

Other acylating agents such as those defined in R₁ above can be used inplace of the 2-thienylacetylchloride shown in the foregoing flowsheet toproduce the corresponding 7-acylamido cephem compounds. In using suchacylating agents it is necessary to avoid the use of acylating agentscontaining substituents which would be affected during the reactions.Thus, amino, carboxy or hydroxy substituents of the acylating agentshould be blocked or protected by groups such as those mentioned aboveand then subsequently removed. Examples of other specific acylatingagents that might be mentioned are phenylacetylchloride,2-furylacetylchloride, thiophenoxyacetylchloride,α-azidophenylacetylchloride, and the like. Alternatively, otheracylating agents such as the anhydrides or mixed anhydrides can beutilized in place of the acid halides. This method of transacylation isindeed a valuable advance in this art since it provides a means ofpreparing cephalosporins containing different 7-acylamido substituentsin place of the aminoadipoylamido group and thereby avoids the need forfirst converting known cephalosporins to the corresponding7-aminocephalosporanic acid compound and then acylating this product. Inaddition to using cephalosporins produced by fermentation as startingmaterials in this process, derivatives of such cephalosporins containingother substituents at 3 in place of the carbamoyloxymethyl or theacetoxymethyl substituent such as 3-substituents of the general formulaCH₂ A defined above can be utilized. Alternatively, other 3-substitutedcephalosporins can be prepared, for example, from the3-acetoxymethyl-7-acylamidocephalosporins, in accordance with methodswell known in this art.

Thus, examples of other cephalosporins having a 7-methoxy or 7-hydrogensubstituent which can be prepared by the above-described processes thatmight be mentioned are shown in the following table:

    ______________________________________                                        7-acylamido substituent                                                                          3-substituent                                              ______________________________________                                         ##STR31##         CH.sub.3                                                    ##STR32##                                                                                        ##STR33##                                                 φSCH.sub.2CONH                                                                                ##STR34##                                                  ##STR35##         CH.sub.2 OCONH.sub.2                                        ##STR36##                                                                                        ##STR37##                                                 CH.sub.3 (CH.sub.2).sub.3 SCH.sub.2CONH                                                          CH.sub.2 OCH.sub.3                                          ##STR38##                                                                                        ##STR39##                                                 ______________________________________                                    

An alternative route for the preparation of the 7-R₁ -7-amino compoundsof formula IX above comprises reacting a 7-amino compound of formula IIabove with an aromatic aldehyde to form an imino adduct, treating thisimino adduct with a defined reagent yielding a 7-R₁ Schiff's base adductand then regenerating the amino moiety. This process is not a part ofthis invention but is disclosed and claimed in a co-pending application,Great Britain Ser. No. 13,008, filed Mar. 16, 1972.

More specifically, this alternative route can be used to preparecompounds having the following formula: ##STR40## wherein A and R₈ areas previously defined, and R₁ is lower alkyl, lower alkoxy, loweralkylthio, lower alkanoyl, lower haloalkoxy, lower haloalkylthio, halo,lower haloalkyl, lower alkanoyloxy, (α-hydroxy) lower alkyl, (α-hydroxy)lower alkenyl, β-substituted ethylene derivatives, allyl, benzyl, cyano,nitroso, carbamoyl, carboloweralkoxy, sulfo, sulfonoyl, loweralkylsulfo,phospho, nitro, carboxy, and dithiocarboxy.

The starting material is the 7-NH₂ compound of formula II above which isreacted with an aromatic aldehyde, preferably one having at least one o-or p-electronegative substituent, selected from the group consisting ofnitro, methyl sulfonyl, cyano, carboxyl, derivatives, and the like. Thepreferred reactant is p-nitrobenzaldehyde.

The starting material and the aromatic aldehyde are mixed together inapproximately equimolar amounts in an inert solvent. Suitable solventsare dioxane, acetonitrile, dimethylformamide, dimethylsulfoxide,benzene, toluene, and the like. The aldehyde can be employed in amolecular excess if desired. The reaction proceeds readily attemperatures ranging from ambient to reflux temperature of the solvent.Since this condensation is an equilibrium reaction and since water isone of the products of the reaction, water is removed from activeparticipation in further reactions by any of a number of usual methods,including azeotropic distillation, molecular sieves, or borate esters.The particular method is dependent upon the exact parameters of thereaction. The reaction is terminated by evaporation of the solvent. Theimino derivative is then recovered and used in the next step.

The latter involves the substitution of the R₁ group at the carbon atomadjacent to the imino nitrogen. This reaction takes place in thepresence of an inert solvent, such as those listed above, and in theadditional presence of an organic or inorganic base. It is preferred touse organic bases, such as tertiary amines or pyridine. A specifictertiary amine which is preferred is diisopropylethylamine, although anytertiary lower alkylamine can be used. Inorganic bases, such as NaH,NaOH, KOH, carbonate, or bicarbonate salts, etc. can also be employed.For instance, the reaction can be conducted in "soft glass" whichcontains enough soluble inorganic base to catalyze the reaction.

The specific reactant which is employed in the reaction with the iminocompound to result in the chosen R₁ group obviously depends on the R₁group desired.

The following is of value in defining each reactant in terms of thefinal R₁ group.

    ______________________________________                                        Reactant             R.sub.1                                                  ______________________________________                                        1.   lower alkyl sulfate or halide                                                                     loweralkyl                                           2.   loweralkanoyl halide                                                                              loweralkanoyl                                        3.   loweralkyl peroxide loweralkoxy                                          4.   haloloweralkyl peroxide                                                                           lowerhaloalkoxy                                      5.   loweralkyl disulfide                                                                              loweralkylthio                                       6.   haloloweralkyl disulfide                                                                          lowerhaloalkylthio                                   7.   tertbutylhypohalite or                                                        perhalomethylhypohalite                                                                           halo                                                 8.   haloloweralkane     haloloweralkyl                                       9.   loweralkanoyl peroxide                                                                            loweralkanoyloxy                                     10.  formaldehyde or loweralkyl-                                                   aldehyde            (α-hydroxy) loweralkyl                         11.  reactive loweralkyl ketone                                                                        (α-hydroxy) branched-                                                   loweralkyl                                           12.  reactive ethylene derivatives                                                                     (β-substituted) ethyl                           13.  allyl halide        allyl                                                14.  benzyl halide       benzyl                                               15.  cyanogen bromide    cyano                                                16.  nitrosyl halide     nitroso                                              17.  carbamoyl halide    carbamoyl                                            18.  loweralkylhalo formate                                                                            carboloweralkoxy                                     19.  sulfuryl chloride   sulfo                                                20.  sulfamoylchloride   sulfamoyl                                            21.  loweralkylsulfonyl halide                                                                         loweralkylsulfo                                      22.  phosphorus oxychloride                                                                            phospho                                              23.  acetonecyanhydrinnitrate                                                                          nitro                                                24.  carbondioxide       carboxy                                              25.  carbondisulfide     dithiocarboxy                                        ______________________________________                                    

"(α-Hydroxy)loweralkyl" is used to mean a group of the formula ##STR41##wherein R is hydrogen or alkyl having 1-6 carbon atoms.

"Reactive loweralkyl ketone" is used to mean a ketone of the formula##STR42## wherein one of R' or R" is a halogenated loweralkyl group, thehalogen-substituted carbon being adjacent to the carbonyl function; orone of R' or R" is an alkyl carbonyl group. The carbonyl being adjacentto the carbonyl of the ketone; the other of R' or R" is loweralkyl.Thus, to illustrate, one type of "reactive loweralkyl ketone" is:##STR43## wherein X is halo, X" is halo or hydrogen, and X' is halo,hydrogen, or loweralkyl; and R" is loweralkyl.

The other type is ##STR44##

R" is hydrogen or loweralkyl, and wherein R" is loweralkyl,haloloweralkyl, loweralkoxy, or lowerhaloalkoxy.

"(α-Hydroxy)branchedloweralkyl" means a group of the formula ##STR45##wherein X, X', X", R', and R" are as defined above.

"Reactive ethylene derivative" is used to mean an ethylenicallyunsaturated compound which is activated by the presence of one or morestrong electron withdrawing groups. For example, compounds of theformula CH₂ ═CHY where Y is ##STR46## and the like are included.

The term "(β-substituted)ethyl" is employed to mean the following group

    --CH.sub.2 --CH.sub.2 Y

wherein Y is the same as defined as above.

Following the reaction between the imino compound and the reactant toform the novel 7-R' compounds, the imino moiety is regenerated to amino.

This regeneration is effected by aminolysis or hydrazinolysis, in thepresence of a catalytic amount of acid. Preferably, anilinehydrochloride is employed which serves both as a source of amine andacid. When hydrazine or hydrazine derivatives such as phenylhydrazine,2,4-dinitrophenylhydrazine, and the like are used, acid is added. Otherhydrazines or amines can be employed. Preferred media are theloweralkanols, such as methanol, ethanol, and the like. The usual acidsor bases can be employed. For instance, hydrochloric p-toluene sulfonicacid or aniline can be used. The only limitation is that no undesiredhydrolysis or ring damage occur.

The 7-R₁ -7-aminocephalosporanic acid and 7-R₁ -7-aminodecephalosporanicacid esters of formula IX above prepared in this way can then beconverted to the cephalosporin compounds following the proceduresdescribed above.

The step of acylating the 7-amino compounds of formula IX above iseffected by reacting the amine compound with the acyl acid in thepresence of an activating agent such as dicyclohexyldiimide, with theacyl anhydride, with an acyl halide such as the acid chloride, or withan activated ester of the acid such as the p-nitrophenyl ester. In theprocess of reductively acylating the 7-azido compounds of formula VIIIabove, the reductive acylation is preferably effected in the presence ofthe acyl anhydride.

The following examples are given for the purpose of illustration and notby way of limitation.

EXAMPLE 1 A. Benzyl 7-diazocephalosporanate

A mixture of 10 g. sodium nitrite, 4.5 g. benzyl 7-aminocephalosporanatep-toluenesulfonic acid salt, 300 ml. methylene chloride and 300 ml.water/ice is shaken in a separatory funnel. p-Toluenesulfonic acidmonohydrate (1.6 g.) is added in three portions during 20 minutes. Theseparatory funnel is shaken vigorously during this time. The methylenechloride layer is separated, dried over sodium sulfate and evaporatedunder reduced pressure to 40 ml.

B. Benzyl 7-azido-7-bromocephalosporanate

To the solution of benzyl 7-diazocephalosporanate in 40 ml. of methylenechloride is added 40 ml. of nitromethane, and the resulting solution iscooled in an ice bath. To this solution is added 80 ml. oftriethylammonium azide in methylene chloride prepared as describedbelow. To this reaction mixture is then added 40 ml. of methylenechloride containing bromine azide prepared as described below with goodstirring. After the gas evolution ceases, 200 ml. of 0.1 N sodiumthiosulfate is added and the mixture is shaken vigorously. The organiclayer is then separated and 200 ml. of water added. To this mixture isthen added solid sodium bicarbonate in small portions until the aqueousphase remains at pH 7 after shaking. The organic layer is thenseparated, dried with magnesium sulfate and evaporated under reducedpressure to afford 4.8 g. of crude benzyl7-azido-7-bromocephalosporanate in the form of a brown oil. The productis purified further by chromatography on 120 g. of silica gel usinghexane/benzene in 1:1 and 1:3 ratio to elute the product. The fractionscontaining clean product are combined and evaporated to give benzyl7-azido-7-bromocephalosporanate. IR (liq. film): 4.7μ (azide), 5.60μ(β-lactam carbonyl), 5.75μ (esters). NMR_(CDCl).sbsb.3^(TMS) (100 MHz):##STR47## 3.42δ (AB, 2H, S--CH₂ --), 4.68 and 4.71δ (singlets, C-6hydrogens). Thin layer chromatography Rf of 0.70 on silica gel G using1% methanol in chloroform.

The solution of triethylammonium azide is prepared by mixing 6.0 g. ofsodium azide, 20 ml. of water and 50 ml. of methylene chloride, coolingthis reaction mixture to 0° C. and adding 6 ml. of concentrated sulfuricacid. The resulting reaction mixture is allowed to stir for 10 minutes,the layers are separated and the aqueous layer washed with a smallamount of methylene chloride which is added to the previously separatedmethylene chloride phase. After drying with calcium chloride, themethylene chloride solution is neutralized to pH 7 with triethylamineand the final volume is adjusted to 100 ml. with methylene chloride.

The solution of bromine azide is prepared by cooling a mixtureconsisting of 26 g. of sodium azide, 80 ml. of methylene chloride and6.4 g. of bromine to 0° C., adding 20 ml. of concentrated hydrochloricacid, allowing the reaction mixture to stir in an ice bath for 3 hours,separating the organic phase, washing the aqueous phase with a smallquantity of methylene chloride which is added to the separated organicphase, and adjusting the volume to 100 ml. with methylene chloride.

C. Benzyl 7-azido-7-methoxycephalosporanate

A solution of benzyl 7-azido-7-bromocephalosporanate in methanol istreated with one equivalent of dry silver fluoroborate. A buff coloredprecipitate forms rapidly. The mixture is stirred at 22° C. for 23/4hours. The solid is removed by filtration and the filtrate is evaporatedto afford crude benzyl 7-azido-7-methoxycephalosporanate. The crudeproduct is chromatographed on silica gel using 2% chloroform in benzene.The desired product has NMR_(CDCl).sbsb.3^(TMS) (Partial, 100 MHz##STR48## 3.60δ (S, 3H, OCH₃). IR (liq. film): strong absorption at4.70μ (azide), 5.60μ (β-lactam), 6.76μ (esters). Thin layerchromatography Rf of 0.65 on silica gel G with 1% methanol inchloroform.

D. Benzyl 7-acetamido-7-methoxycephalosporanate

A mixture of 70.5 mg. benzyl 7-azido-7-methoxycephalosporanate, 69.5 mg.of platinum oxide and 5.0 ml. acetic anhydride is hydrogenated atatmospheric pressure for 16 hours. The solvent is evaporated underreduced pressure, and the crude product is chromatographed on 12 g.silica gel using chloroform and chloroform with 1-5% methanol. Crudebenzyl 7-acetamido-7-methoxycephalosporanate elutes with 1% methanol inchloroform. The product has IR (liq. film) 3.0μ (NH), 5.60μ (β-lactamcarbonyl). 5.75μ (esters), 5.93 and 6.60μ (amide) and no absorption at4.7μ (azide). NMR_(CDCl).sbsb.3^(TMS) (partial, 100 MHz): ##STR49##3.50δ (singlet, OCH₃), 5.20δ (singlet, C-6 hydrogen). Thin layerchromatography Rf of 0.40 on silica gel G with 5% methanol inchloroform.

E. Potassium 7-acetamido-7-methoxycephalosporanate

A solution of benzyl 7-acetamido-7-methoxycephalosporanate (25 mg.) in 3ml. of 1:1 aqueous methanol is hydrogenated using 25 mg. of 10% Pd/Ccatalyst at 40 p.s.i. of hydrogen for 1 hour. The mixture is filteredand the pH of the filtrate adjusted to 8 with potassium bicarbonate. Theaqueous solution is lyophilized to give potassium7-acetamido-7-methoxycephalosporanate.

EXAMPLE 2 A. Benzhydryl 7-aminocephalosporanate

To a slurry of 6.8 g. (0.025 mole) of 7-aminocephalosporanic acid in 300ml. of peroxide-free dioxane at room temperature is added with stirring4.3 g. (0.022 mole) of p-toluenesulfonic acid monohydrate. The clearsolution is concentrated in vacuo and flushed twice with dioxane.

The residue is dissolved in 300 ml. of dioxane at room temperature, anda solution of 10 g. (0.05 mole) of diphenyldiazomethane in 25 ml. ofdioxane is added dropwise over 15 minutes. The wine-colored solution isstirred for an additional 30 minutes, then 25 ml. MEOH is added todestroy the excess φ₂ CN₂. The mixture is concentrated in vacuo and theresidue partitioned between 200 cc. CH₂ Cl₂ and 200 ml. water containing10 g. K₂ HPO₄ (pH 8.5). The organic phase is washed with water, driedover Na₂ SO₄ and concentrated in vacuo to yield an oil.

The oil is stirred with 100 ml. of ether for 1 hour. The precipitate isfiltered, washed with ether and dried to constant weight 4.7 g. (43%).m.p.=126°-128° C. Analysis calculated: C, 63.0; H, 5.01; N, 6.37. Found:C, 62.7; H, 5.18; N, 5.18. IR in CHCl₃ is 5.6μ (β-lactam C═O) and 5.8μ(ester C═O). NMR in CDCl₃ is 1.85 δ(singlet, NH₂); ##STR50## 3.45δ(doublet, CH₂ S); 4.8 δ(singlet, CH₂ OAC); 4.7 δ(doublet, C₆ H); 4.9δ(doublet, C₇ H); ##STR51## and 7.4 δ(singlet, phenyl).

B. Benzhydryl 7-diazocephalosporanate

To a stirring mixture of 1.6 g. of NaNO₂, 30 ml. of water and 40 ml. ofCH₂ Cl₂ at 0° C. is added 880 mg. (0.002 mole) of ester followed by theaddition of a solution of 760 mg. (0.004 mole) of p-toluenesulfonic acidin 5 ml. water over a few minutes. The mixture is stirred at 0° C. for20 minutes, then the organic phase is cut away, washed with 1×10 cc. icewater, dried over Na₂ SO₄ at 0° C., filtered and concentrated in vacuoat room temperature to yield 900 mg. of a glass. IR is 4.8μ (strongN═N), 5.6μ (β-lactam C═O) and 5.8μ (ester C-O). NMR in CDCl₃ is##STR52## 3.4 δ(doublet, CH₂ S); 4.8 δ(singlet, CH₂ OAC); 5.6 δ(singlet,C₆ H); ##STR53## and 7.4 δ(singlet, phenyl).

C. Benzhydryl 7-bromo-7-azidocephalosporanate

To a solution of 900 mg. of benzhydryl 7-diazocephalosporanate in 20 ml.CH₂ Cl₂ and 10 ml. CH₃ NO₂ at 0°-10° C. is added all at once the Et₃ N⁺NH₃ ⁻ followed by the BrN₃ solution, then 50 ml. of water is addedfollowed by the addition of solid NaHCO₃ to pH 8.

The organic layer is separated and extracted with 2×20 ml. water, driedover Na₂ SO₄ and concentrated in vacuo to yield 900 mg. (83%).

The NMR fits the structure. Thin layer chromatography on silica gel withCHCl₃ shows a major spot at Rf 0.2. Chromatography of 900 mg. crudeproduct on 25 g. silica gel with CHCl₃ gives 400 mg. (39%) single spotmaterial as an oil.

IR in CHCl₃ is 4.72μ (N₃), 5.56μ (β-lactam C═O) and 5.75μ (ester C═O).NMR in CDCl₃ is ##STR54## 3.38 δ(CH₂ S); 4.7 δ(singlet C₆ HO); 4.94δ(CH₂ --O) ##STR55## and 7.4 δ(singlet, phenyl).

Preparation of BrN₃ Solution

To 8 ml. of CH₂ Cl₂ at 0° C. is added 2.66 g. (0.04 mole) of NaN₃followed by 0.65 g. (0.0042 mole) of bromine. To this stirring mixtureat 0° C. is added dropwise 2 ml. of concentrated hydrochloric acid. Themixture is stirred for 3 hours at 0° C.

The organic layer is decanted and the aqueous layer extracted with 1×5ml. of CH₂ Cl₂. The combined organic phase is stored at -10° C.

Preparation of Et₃ N_(H) ⁺ N₃ ⁻ Solution

To a slurry of 1.5 g. of NaN₃ in 5 ml. water and 10 ml. CH₂ Cl₂ at -10°C. is added dropwise at -10° C. to 0° C. 4 ml. of 50% H₂ SO₄. Theorganic phase is poured off the aqueous paste, and the aqueous extractwashed with 1×5 cc. CH₂ Cl₂. The combined organic phase is dried overCaCl₂. The decanted HN₃ solution is brought to pH 7 with Et₃ N andstored at -10° C.

D. Benzhydryl 7-methoxy-7-azidocephalosporanate

To a solution of 400 mg. (0.00072 mole) of bromoazide in 30 ml. methanolis added 150 mg. (0.0008 mole) of AgBF₄. The mixture is stirred in thedark for 21/2 hours.

The mixture is concentrated in vacuo and the residue taken up in 50 ml.of CH₂ Cl₂ filtered. The filtrate is extracted twice with saturatedNaHCO₃ solution, twice with water, dried over anhydrous MgSO₄ andconcentrated in vacuo to yield 300 mg. (83%) of crystals. m.p.=145°-148°C.

IR in CHCl₃ is 4.72μ (N₃ band), 5.6μ (β-lactam) and 5.75μ (ester C═O).NMR is ##STR56## 3.4 δ(CH₂ S); 3.6 δ(singlet, OCH₃); 4.88 δ(singlet, C₆H); 4.9 δ(CH₂ O); ##STR57## and 7.4 δ(singlet, phenyl).

Analysis calculated: C, 58.4; H, 4.45, N, 11.3; S, 6.5. Found: C, 58.56;H, 4.65; N, 11.30; S, 5.70.

EXAMPLE 3 A. Benzhydryl 7-methoxy-7-aminocephalosporanate

1.0 g. of benzhydryl 7-azido-7-methoxycephalosporanate is dissolved in100 ml. of dioxane. 1.0 g. of platinum oxide is added and the reactionmixture stirred under hydrogen at atmospheric pressure for 1 hour.Another 1.0 g. quantity of platinum oxide is added and the reactionmixture is again placed under hydrogen and stirred for 3 hours until theazide is completely reacted as determined by infrared analysis ofaliquots. The solvent is removed under reduced pressure and the residuetaken up in 50 ml. of chloroform and filtered through silica gel G inchloroform in a 60 ml. sintered glass funnel. The material is elutedwith chloroform until 200 ml. of chloroform has been collected. Thechloroform is removed under reduced pressure affording 0.632 g. ofbenzhydryl 7-methoxy-7-aminocephalosporanate, which is acylated directlywithout further purification. The starting compound is prepared usingthe procedures described in Example 1 starting with the benzhydryl esterof 7-aminocephalosporanic acid.

B. Benzhydryl 7-methoxy-7-(2-thienylacetamido)cephalosporanate

0.632 g. of benzhydryl 7-methoxy-7-aminocephalosporanate is taken up in25 ml. of methylene chloride and cooled to 0° C. 0.6 ml. of 2-thienylacetyl chloride is added dropwise over 30 seconds followed by 0.6 ml. ofpyridine 60 seconds later. The reaction mixture is stirred at 0° C. for15 minutes and poured into crushed ice. The mixture is agitated and theorganic layer separated and washed once with 20 ml. of water, once with20 ml. of 5% sodium bicarbonate and once again with 20 ml. of water. Themethylene chloride is dried and evaporated to dryness affording 1.417 g.of crude product. This material is placed on a column of 60 g. of silicagel under benzene and the column is eluted with benzene, taking 100 ml.fractions followed by 300 ml. of methylene chloride/benzene (1:1) in 3fractions, and 500 ml. of methylene chloride in 5 fractions. The productis removed from the column by eluting with 400 ml. of chloroform in 4fractions, affording 0.592 g. This material is taken up in 25 ml. ofmethylene chloride and stirred at room temperature with 20 ml. of asolution of 0.120 g. of sodium bicarbonate in water for 1/2 hour. Thelayers are separated and the organic layer washed with water, dried andevaporated to dryness, affording 0.420 g. of benzhydryl7-methoxy-7-(2-thienylacetamido)cephalosporanate, which shows 1 spot ona thin layer chromatographic plate. ##STR58##

C. Sodium 7-methoxy-7-(2-thienylacetamido)cephalosporanate

0.420 g. of benzhydryl 7-methoxy-7-(2-thienylacetamido)cephalosporanateis dissolved in 3.5 ml. of anisole and treated with 10 ml. oftrifluoroacetic acid at room temperature for 10 minutes. Thetrifluoroacetic acid and anisole are removed under reduced pressuremaintaining the temperature below 40° C., and the residue is taken up in25 ml. of chloroform and treated with 20 ml. of water containing 0.120g. of sodium bicarbonate. The mixture is stirred for 1/2 hour at roomtemperature and the organic phase is separated and washed with water.The combined aqueous phase is washed twice with methylene chloride andlyophilized affording 0.382 g. of sodium7-methoxy-7-(2-thienylacetamido)cephalosporanate as a brownish solid.IR: 5.65μ (β-lactam), 5.91μ (amide carbonyl). NMR (DMSOD₆): 2.65 tau(singlet) and 3.06 tau (doublet) (thienyl protons); 5.04 tau (singlet,6H); ##STR59##

EXAMPLE 47-Methoxy-7-(2-thienylacetamido)-3-desacetoxy-3-pyridiniumcephalosporanicacid thiocyanate

0.100 g. of sodium 7-methoxy-7-(2-thienylacetamido)cephalosporanate isdissolved in 100 μl of water containing 50 μl of pyridine, 5 μl of 85%phosphoric acid and 0.475 g. potassium thiocyanate. The reaction mixtureis stirred at 60° C. for 5 hours and cooled to room temperature. Thereaction mixture is diluted with water to a total volume of 20 ml. andextracted 5 times with 5 ml. portions of chloroform. Chloroform isremoved from the aqueous phase by evaporation under vacuum and theaqueous phase is then cooled to 0° C. and acidified to pH 2. The mixtureis allowed to stand at 0° C. for 2 hours and the precipitated solidremoved by filtration and dried to give 0.015 g. of7-methoxy-7-(2-thienylacetamido)-3-desacetoxy-3-pyridiniumcephalosporanicacid thiocyanate as a pale yellow solid. IR: 4.83μ (CNS⁻), 5.62μ(β-lactam). Rf 0.61 (BAW 3:1:1, on paper).

EXAMPLE 5 A. Benzhydryl7-methoxy-7-(2-thianaphthene-2-acetamido)cephalosporanate

330 mg. (1.57 mm.) of benzothiophene-2-acetyl chloride is added in 1portion to a solution of 330 mg. (0.75 mm.) benzhydryl7-amino-7-methoxycephalosporanate in 10 ml. of methylene chloride at 0°C. 330 μl of dry pyridine is added after 1 minute and the homogeneousreaction mixture stirred for 15 minutes at room temperature and pouredonto 10 ml. of water. The organic layer is washed once with 3 ml. ofcold aqueous 5% sodium bicarbonate and once with 3 ml. of cold water anddried over sodium sulfate. The solvent is removed in vacuo at roomtemperature and the crude product chromatographed on a 2.1 cm. (outsidediameter) column containing 30 g. of silica gel under methylenechloride. All less polar contaminants, including the azide from theprevious step, are eluted with 400 ml. of methylene chloride. The eluantis then changed to chloroform to remove the product. All fractionscontaining the product are combined and washed with 5% sodiumbicarbonate and water. The solution is dried over sodium sulfate andevaporated to dryness in vacuo affording 301 mg. of benzhydryl7-methoxy-7-(2-thianaphthene-2-acetamido)cephalosporanate as a gold oil.IR: 6.65μ (β-lactam), 5.78μ (ester), 5.95μ (amide). ##STR60## TLC:silica gel G, 5% EtOAc/CH₂ Cl₂ Rf=0.37.

B. Sodium 7-methoxy-7-(2-thianaphthene-2-acetamido)cephalosporanate

2.0 ml. of anisole and 5.9 ml. of trifluroacetic acid are combined andadded to 300 mg. of the benzhydryl7-methoxy-7-(2-thianaphthene-2-acetamido)cephalosporanate, and thereaction mixture is stirred for 10 minutes at room temperature. Excessanisole and trifluoroacetic acid are removed in vacuo and residualanisole and trifluoroacetic acid are removed by placing the flaskdirectly onto a high vacuum pump for 15 minutes. The residual dark oilis taken up in 15 ml. of a benzene/ethyl ether mixture (1:1). A smallamount of insoluble gum is dissolved by the addition of 5 ml. of a 5%sodium bicarbonate solution in water. The organic layer is washed withwater and the combined aqueous layers are lyophilized to give 185 mg. ofsodium 7-methoxy-7-(2-thianaphthene-2-acetamido)cephalosporanate as apale yellow powder. NMR: 6.18 tau (--CH₂ CNH--), 8.13 tau (--O═C--CH₃),6.52 tau (--OCH₃).

EXAMPLE 6 Benzhydryl 7-amino-7-methoxycephalosporanate

500 mg. of platimum oxide is added to a solution of 500 mg. (1.07 mm.)of benzhydryl 7-azido-7-methoxycephalosporanate in 50 ml. of p-dioxanein a 250 ml. round bottom flask. The reaction vessel is placed underhydrogen at room temperature and atmospheric pressure with vigorousmagnetic stirring. After 1 hour, 500 mg. of fresh platinum oxide isadded and the reaction continued under the same conditions for anadditional 3 hours. The dioxane is removed in vacuo at room temperatureand the residue taken up in 5 ml. of chloroform. The catalyst is removedby passing the mixture through 15 g. of silica gel G packed in asintered glass funnel. The product is eluted with 400 ml. of chloroformusing a vacuum. The chloroform is evaporated in vacuo affording 300 mg.of benzhydryl 7-amino-7-methoxycephalosporanate as a yellow oil.

EXAMPLE 7 A. 7-Methoxy-7-(p-guanidinophenylacetamido)cephalosporanicacid benzhydryl ester hydrochloride

To a solution of 250 mg. of 7-amino-7-methoxycephalosporanic acidbenzhydryl ester, dissolved in 1.5 ml. of dry dimethylformamide, isadded in 1 portion with cooling and stirring 0.15 g. ofp-guanidinophenyl acetyl chloride and 0.15 ml. of pyridine. The reactionmixture is stirred at 5° C. for 5 minutes and at room temperature for 10minutes. The solution is diluted with 15 ml. of methylene chloride andextracted 3 times with 15 ml. of water. The methylene chloride solutionis dried and evaporated to dryness in vacuo and the residue ischromatographed through 20 g. of silica gel. Elution with chloroform,followed by an ethanol/chloroform mixture (1:1), affords 100 mg. of7-methoxy-7-(p-guanidinophenylacetamido)cephalosporanic acid benzhydrylester hydrochloride. Thin layer chromatography on silica gel using 1:1ethanol/chloroform shows a single spot with a Rf of 0.6. IR: 6.5μ(lactam), 5.8μ (ester).

B. 7-Methoxy-7-(p-guanidinophenylacetamido)cephalosporanic acid

A solution of 88 mg. of the above ester and 0.7 ml. of anisole dissolvedin 1.8 ml. of trifluoroacetic acid is kept at room temperature for 10minutes. The solution is evaporated on the high vacuum pump for 5minutes and the residue is triturated with ether until it solidifies.After decanting the ether, the solid is stirred with 50 ml. of water andfiltered. The clear filtrate is lyophilized, affording 40 mg. of7-methoxy-7-(p-guanidinophenylacetamido)cephalosporanic acid. Circularpaper chromatography using butanol-acetic acid in water (3:1:1) shows 1spot of Rf 0.25 which gives a positive Sakaguchi test and bioactivityagainst B. subtilis. IR: 5.7μ (ester, shoulder), 5.65μ (lactam).

EXAMPLE 8 Sodium 7-methoxy-7-(2-furylacetamido)cephalosporanate

Benzhydryl 7-methoxy-7-azidocephalosporanate, 1.00 g., is hydrogenatedin 100 ml. dioxane for 1 hour with 1 g. PtO₂, then for 3 more hours withanother gram of PtO₂. The solvent is removed in vacuo at a temperaturebelow 30° C. The residue is taken up in chloroform and filtered througha bed of about 1 inch of silica gel (thin layer chromatography grade),washing copiously with chloroform; total volume about 500 ml. Removal ofthe chloroform in vacuo affords benzhydryl7-methoxy-7-aminocephalosporanate.

Methylene chloride, 40 ml., is added, and then at 0° C., first 0.7 ml.of furylacetyl chloride and then 1 ml. of pyridine are added. After 25minutes of stirring at 0° C. water is added and stirring continued for afew more minutes. The layers are separated and the organic portionwashed successively with 1% aqueous H₃ PO₄, water, and saturated aqueoussodium bicarbonate. After drying the methylene chloride solution withMgSO₄, filtering and evaporating the solvent, benzhydryl7-methoxy-7-(2-furylacetamido)cephalosporanate is obtained. It ispurified by chromatography on 60 g. neutral silica gel (Brinkmann's70-325 mesh ASTM), and eluted with 4:1 chloroform-ethyl acetate. Its Rfon the column is 0.69-0.45, and on TLC in the same system is found to be0.69-0.57, single spot. Its IR spectrum (CHCl₃ solution) has bands at3.0μ (N-H), 5.61μ (β-lactam), 5.76μ (esters) and 5.9μ (amide). The NMRspectrum in CDCl₃ has bands at 8.05 tau (3H, singlet, --COCH₃), 6.65,6.70 tau (2H, S--CH₂ --), 6.55 tau (3H, singlet, --OCH₃), 6.32 tau (2H,singlet, furyl--CH₂ --), 4.85 5.05, 5.15, 5.35 tau (2H, AB qt., J=14 Hz,--CH₂ OAc), 4.97 tau (1H, singlet, C₆ --H), 3.72 tau (2H, m, furyl β-H),3.09 tau (1H, singlet, --CHφ₂), 2.7 tau (6H, m, phenyl and furyl α--H).

This compound, 0.57 g., is treated at 0° C. for 5 minutes with 0.8 ml.anisole and 4.0 ml. trifluoroacetic acid. The TFA and anisole areremoved below 30° C. in vacuo, and 2 ml. more anisole is added andevaporated as before. The residue is taken up in a few ml. watercontaining 0.1 g. NaHCO₃ and lyophilized to a powder, which is washedcopiously with ether and dried to afford 0.45 g. of sodium7-methoxy-7-(2-furylacetamido)cephalosporanate. Its IR spectrum (CHCl₃solution) has bands at ca. 3.1μ (broad, N--H), 5.67μ (β-lactam), 5.75μ(ester), 5.92μ (amide) and 6.18μ (COONa). Its NMR spectrum in D₂ O hasbands at 7.92 tau (3H, --COCH₃), 6.60, 6.66 tau (2H, S--CH₂ --), 6.47tau (3H, --OCH₃), 6.20 tau (2H, furyl-CH₂ --), 5.38 tau (HDO), 5.13 tau(2H, --CH₂ OAc), 4.88 tau (1H, C₆ --H), 3.63 tau (2H, furyl β--H), 2.53tau (1H, furyl α-H). The UV spectrum in pH 7 buffer has λmax. 263 nm,E%=141. Its Rf on TLC (silica gel, acetone-AcOH 9:1) is 0.68.

EXAMPLE 9 A. Benzhydryl 7-methoxy-7-tetrazolylacetamidocephalosporanate

To a solution of 1.17 g. benzhydryl 7-amino-7-methoxycephalosporanate in100 ml. methylene chloride cooled at 0°-5° C. in an ice bath is added1.78 ml. of pyridine with stirring followed by a cooled solution of 1.17g. tetrazolylacetyl chloride in 100 ml. methylene chloride. The mixtureis allowed to react for 10 minutes at 0°-5° C. It is then shaken with200 ml. of pH 2 phosphate buffer. The methylene chloride layer is driedand evaporated. The crude product, 1.2 g., is eluted through 48 g. ofsilica gel, using chloroform as an eluant. The desired product is elutedwith 2% methanol/chloroform yielding 450 mg. of benzhydryl7-methoxy-7-tetrazolylacetamidocephalosporanate. NMR: 8.0 tau (acetylsinglet), 6.53 tau (SCH₂), 4.84 tau (6H, singlet), 3.0 tau (singlet, CHof benzhydryl), 2.6 tau (singlet, aromatic), 1.1 tau (tetrazole H). TLCon silica gel in 5 % methanol/chloroform Rf=0.28.

B. Sodium-7-methoxy-7-tetrazolylacetamidocephalosporanate

A mixture of 680 mg. of benzhydryl7-methoxy-7-tetrazolylacetamidocephalosporanate, 4.4 ml. anisole and12.2 ml. trifluoroacetic acid is stirred at room temperature for 8minutes. The excess acid is evaporated under reduced pressure and theresidue is flushed twice with carbon tetrachloride and then thrice withhexane. The solid residue is dissolved in ethylacetate, adjusted to pH5.8 with sodium bicarbonate solution, extracted with water and freezedried to yield 420 mg. of sodium7-methoxy-7-tetrazolylacetamidocephalosporanate. UVλ_(max).^(H).sbsp.2^(O) 265 E% 104.

EXAMPLE 10 Sodium7-methoxy-7-[1(1H)-tetrazolylacetamido]-3-(5-methyl-1,3,4-thiadiazolethiomethyl)cepham-4-carboxylate

A mixture of 420 mg. sodium7-methoxy-7-tetrazolylacetamidocephalosporanate, 168 mg.2-methyl-5-mercapto-1,3,4-thiadiazole and 16.8 ml. phosphate buffer (pH6.4) is heated on the steam bath for 1/2 hour. The reaction mixture isadjusted to pH 4.85 and extracted with 25 ml. ethylacetate. The aqueouslayer is adjusted to pH 1.8 with 2.5 N hydrochloric acid solution andextracted with ethylacetate. Ethylacetate is removed under reducedpressure and the residue is flushed thrice with ethanol to remove tracesof ethylacetate. Then it is taken into ethanol and water, adjusted to pH6.5 with sodium bicarbonate solution, treated with charcoal andfiltered. Ethanol is evaporated and the aqueous layer is freeze dried toyield 210 mg. of sodium7-methoxy-7-[1(1H)-tetrazolylacetamido]-3-(5-methyl-1,3,4-thiadiazolethiomethyl)cephem-4-carboxylate.UV λ_(max).^(H).sbsp.2^(O) 273 E% 155. NMR: 7.28 tau (singlet, methyl ofthiadiazole), 6.42 tau (singlet of methoxy group), 0.74 tau (singlet,tetrazole H).

EXAMPLE 11 A. Benzhydryl 7-aminodesacetoxycephalosporanate

To a mixture of 11.0 g. (0.0514 mole) of7-aminodesacetoxycephalosporanic acid in 1.5 liter of water in a 5 liter3-necked flask fitted with mechanical stirrer and dropping funnel isadded 3.5 g. of boron trifluoride in 80 ml. of dioxane. The mixture isstirred for 1 hour (pH 2.2), 1.5 liter acetone is added, and the mixtureis stirred for 10 minutes (pH 2.4).

Diphenyldiazomethane (31.4 g., 0.162 mole) in 85 ml. acetone is addeddropwise with good stirring during 4 hours, during which time the slurrybecomes thinner. The mixture is filtered and the solid is air dried andwashed well with chloroform to afford 3.8 g. recovered startingmaterial.

This 3.8 g. of starting material is recycled using 750 ml. water, 1.2 g.of BF₃ in 30 ml. dioxane, 750 ml. of acetone and 11 g. Ph₂ CN₂ in 30 ml.acetone as described above.

Filtrates from both reactions are evaporated under reduced pressure toremove acetone. The aqueous residues are extracted with chloroform(3×250 ml. and 3×150 ml.). Drying of the chloroform extracts withmagnesium sulfate and evaporating affords about 45 g. of crude product,which is chromatographed on 125 g. silica gel using chloroform andchloroform-methanol (1-3%) mixtures as eluant. Fractions 3-8 aretriturated individually with ether. Crystals are obtained from fractions4-7: crop 1, m.p. 133°-143° C., 5.58 g.; crop 2, m.p. 95°-125° C., 0.52g.

Filtrates and adjacent fractions from above chromatography are combinedand rechromatographed as before. Fractions 4-12 are triturated withether. Fraction 9 is crystallized well to give crop 3, m.p. 133°-142°C., 1.47 g.

Combined yield of benzhydryl 7-aminodesacetoxycephalosporanate is 7.57g. Starting material is recovered from the aqueous phase byconcentrating in vacuo to remove water and adjusting pH to 3.7. Startingmaterial is precipitated on stirring. After filtering, washing theprecipitate with water and acetone, and drying, 3.19 g. of recoveredstarting material is obtained.

Benzhydryl 7-amino-3-desacetoxycephalosporanate, m.p. 146°-150° C. ischaracterized by IR, NMR and elemental analysis. Calculated for C₂₁ H₂₀N₂ O₃ S: C, 66.30; H, 5.30; N, 7.36. Found: C, 66.24; H, 5.55; N, 7.07.

The 7-aminodesacetoxycephalosporanic acid used as the starting materialis prepared by the hydrogenolysis of 7-aminocephalosporanic acid usingpalladium-on-barium sulfate catalyst in accordance with procedures knownin this art.

D. Benzhydryl 7-azido-7-bromo-3-desacetoxycephalosporanate

A mixture of 7.57 g. benzhydryl 7-aminodesacetoxycephalosporanate, 2.74g. sodium nitrite, 14.7 ml. 2 N sulfuric acid, 400 ml. water and 400 ml.methylene chloride is stirred for 1 hour in a stoppered, ice cold flask.The layers are separated while cold. The aqueous layer is washed withmethylene chloride and the combined CH₂ Cl₂ layers are dried withmagnesium sulfate and evaporated under reduced pressure at ≦30° C. toabout 125 ml. of solution of the benzhydryl 7-diazocephalosporanate.

During the time the above reaction mixture is stirring, triethylammoniumazide and bromine azide are prepared. A mixture of 11.7 g. sodium azide,9.7 ml. concentrated sulfuric acid diluted to 40 ml. with water, and 190ml. of methylene chloride are stirred for 30 minutes in a closed systemin an ice bath. The layers are separated while cold, and the aqueousresidue is washed with 10 ml. of cold methylene chloride. The CH₂ Cl₂layers are combined, dried with MgSO₄ while kept in an ice bath, anddivided into two equal parts. To one is added 4.9 ml. triethylamine; tothe other 4.93 g. N-bromosuccinimide. Both solutions are stored in anice bath until use.

The 7-diazo compound in about 125 ml. of methylene chloride in a 500 ml.round bottomed flask protected with calcium chloride tube and stirredmagnetically is cooled in a solid CO₂ /acetone bath to -40° C. (internaltemperature). The bath temperature is kept at -40° C. to -50° C. duringreaction. The triethylammonium azide solution is added all at once. Thebromine azide solution is added during 5 minutes at -30° C. to -25° C.The flask is removed from the bath and allowed to come to 0° C. during20 minutes.

10 g. of disodium hydrogen phosphate in 300 ml. water is mixed with thereaction mixture and the layers separated. The methylene chloride layeris dried with magnesium sulfate and evaporated to give 10.3 g. crudeproduct, which is chromatographed on 125 g. of silica gel using benzeneas eluant. Fractions are collected when the yellow color approaches thebottom of the column: fraction 1, 3.8 g.; fraction 2, 1.3 g.; totalyield=5.1 g. Both fractions crystallize immediately.

An analytical sample of benzhydryl7-azido-7-bromo-3-desacetoxycephalosporanate, m.p. 122° C. ischaracterized by IR, NMR and elemental analysis. Calculated for C₁₉ H₁₇N₄ O₃ S: C, 51.97; H, 3.53; N, 11.54. Found: C, 52.23; H, 3.59; N,11.63.

C. Benzhydryl 7-azido-7-methoxy-3-desacetoxycephalosporanate

A solution of 5.1 g. (10.5 mmoles) of benzhydryl7-azido-7-bromo-3-desacetoxycephalosporanate in 50 ml. methylenechloride and 200 ml. methanol is prepared. Pyridine (0.844 ml., 10.5mmole) and 2.084 g. silver fluoroborate (10.7 mmole) in 10 ml. methanolare added and the reaction mixture is stirred at 22° C. for 16 1/2hours. The mixture is filtered and the filtrate is evaporated underreduced pressure. The residue is chromatographed on 125 g. of silica gelusing benzene as eluant. Fraction 1 is collected when yellow color nearsbottom of the column. Fractions 2-9 are combined and triturated withmethanol. The methoxy azide crystallizes, m.p. 115°-117° C., 3.156 g.and is characterized by IR, NMR and elemental analysis. Calculated forC₂₂ H₂₀ N₄ O₄ S: C, 60.54; H, 4.62; N, 12.84. Found: C, 60.42; H, 4.36;N, 12.93.

D. Benzhydryl7-(D-α-azidophenylacetamido)-7-methoxy-3-desacetoxycephalosporanate

A mixture of 1.51 g. of benzhydryl7-azido-7-methoxy-3-desacetoxycephalosporanate, 150 ml. dioxane and 1.5g. platinum oxide is stirred under hydrogen for 1 hour. Another 1.5 g.catalyst is added and the mixture is hydrogenated for another hour. Thereaction mixture is evaporated under diminished pressure (≦35° C.). Theresidue is taken up in chloroform and passed through silicagel-diatomaceous earth (1:1) in a 120 ml. sintered funnel. About 500 ml.of eluant is collected. The filtrate is evaporated and the process isrepeated in a 60 ml. sintered funnel.

The filtrate (about 500 ml.) is evaporated under reduced pressure andthe residue is dried with nitrogen to give about 2 g. of benzhydryl7-amino-7-methoxydesacetoxycephalosporanate.

The benzhydryl 7-amino-7-methoxydesacetoxycephalosporanate is taken upin 50 ml. of methylene chloride and stirred magnetically in an ice bath.To the solution is added 1.65 g. D-α-azidophenylacetyl chloride. After 3minutes 1.4 ml. of pyridine is added.

The solution is stirred in an ice bath for 25 minutes, poured into 50ml. ice water and the layers separated. The methylene chloride layer iswashed with 40 ml. of dilute aqueous sodium bicarbonate and 50 ml. ofwater and dried overnight in a refrigerator with magnesium sulfate. Themixture is filtered, evaporated and dried to give 2.0 g. of product. Theproduct is chromatographed on 100 g. of silica gel using benzene andbenzene/chloroform mixtures as eluant. Fractions 16-20(benzene/chloroform 1:3) contain the product, benzhydryl7-(D-α-azidophenylacetamido)-7-methoxy-3-desacetoxycephalosporanate (834mg.), which is characterized by IR and NMR.

E. 7-(D-α-azidophenylacetamido)-7-methoxy-3-desacetoxycephalosporanicacid

A solution of 708.4 mg. of benzhydryl7-(D-α-azidophenylacetamido)-7-methoxy-3-desacetoxycephalosporanate in 2ml. anisole is cooled in an ice bath and 8 ml. of trifluoroacetic acidadded. The reaction solution is kept at 0° C. for 10 minutes withoccasional swirling. The solution is evaporated under reduced pressureand flushed and evaporated twice with anisole. The residue is taken upin 30 ml. methylene chloride and extracted with 3×5 ml. saturatedaqueous sodium bicarbonate solution. The combined aqueous phase iswashed with 2×5 ml. of methylene chloride and the pH adjusted to 1.8with 2.5 N HCl and then extracted with 4×10 ml. ethylacetate. Theextracts are dried with magnesium sulfate and evaporated under reducedpressure at ≦45° C., then put directly onto a pump. The productobtained, (487.4 mg.), is characterized by IR and NMR.

EXAMPLE 127-(D-α-aminophenylacetamido)-7-methoxy-3-desacetoxycephalosporanic acid

To an ice-cooled solution of 487.4 mg. of7-(D-α-azidophenylacetamido)-7-methoxy-3-desacetoxycephalosporanic acidin 6 ml. acetic acid and 9 ml. water is added 3.1 g. of powdered zinc.The mixture is stirred in an ice bath for 10 minutes and then filtered.The solid is washed with 40 ml. of water. The combined aqueous filtratesare saturated with hydrogen sulfide. After filtration the filtrate islyophilized to afford 0.5 g. crude product. This crude product isdissolved in water and relyophilized twice to give 400 mg. of product,which is characterized by IR, NMR, electrophoresis mobility, reactionwith ninhydrin and UV (λmax. 262.5 mμ, ε=5770).

EXAMPLE 13 A. Benzhydryl7-(D-α-azido-2-phenylacetamido)-7-methoxycephalosporanate

To a solution of 1 g. of benzhydryl 7-amino-7-methoxycephalosporanate in25 ml. of methylene chloride at 0° C. is added 1.1 g. ofD-α-azidophenylacetyl chloride in 15 ml. of methylene chloride followedby 1 ml. of pyridine. After 15 minutes stirring at 0° C., the mixture isextracted with 2×5 ml. of cold water, 3×5 ml. of 1% aqueous phosphoricacid, 3×5 ml. of saturated aqueous sodium bicarbonate solution and 2×5ml. of water. The methylene chloride solution is dried over magnesiumsulfate, filtered, and concentrated under reduced pressure to afford 1.1g. of benzhydryl7-(D-α-azido-2-phenylacetamido)-7-methoxycephalosporanate in the form ofan oil. This product is chromatographed on 60 g. of neutral silica geland the product eluted with chloroform. Evaporation of the solventaffords 600 mg. of product whose Rf on the column is 0.069-0.047, and onTLC (silica gel, CHCl₃) is 0.25, single spot. Its IR spectrum (CHCl₃solution) has bands at 3.0μ (N--H), 4.74μ (azide), 5.62μ (β-lactam),5.76μ (ester) and 5.88μ (amide). The NMR spectrum in CDCl₃ has bands at8.05 tau (3H, singlet, --COCH₃), 6.65 tau (doublet, 2H, S--CH₂ --), 6.55tau (3H, singlet, --OCH₃), 4.85, 5.05, 5.15, 5.35 tau (2H, AB qt., J-14Hz, --CH₂ OAc), 4.97 tau (1H, singlet, C.sub.φ --H), 4.89 tau (1H,singlet, φ(N₃)C--H), 3.09 tau (1H, singlet, --CHφ₂), 2.65, 2.75 tau(15H, phenyls).

D. 7-(D-α-azido-2-phenylacetamido)-7-methoxycephalosporanic acid

The product obtained in A above (600 mg.) is treated for 5 minutes at 0°C. with 1 ml. of anisole and 5 ml. of trifluoroacetic acid. Theresulting reaction mixture is evaporated at 30° C. at 0.1 mm. pressureand then twice treated with anisole and evaporated again. The residue soobtained is dissolved in 25 ml. of methylene chloride and extracted with4×3 ml. of saturated aqueous sodium bicarbonate. The aqueous solution iswashed once with 5 ml. of methylene chloride, adjusted to pH 1.8 with 5%phosphoric acid and extracted with 3×10 ml. of ethylacetate. Theethylacetate solution is dried with magnesium sulfate and evaporated toyield 370 mg. of7-(D-α-azido-2-phenylacetamido)-7-methoxycephalosporanic acid. IRspectrum (CHCl₃): 3-4μ (COOH), 4.74μ (azide), 5.62μ (β-lactam), 5.75μ(ester), 5.85μ (amide), ca. 8μ (acid C═O). NMR spectrum (CDCl₃): 7.92tau (3H, singlet, --COCH₃), 6.65 tau (2H, doublet, S--CH₂ --), 6.51 tau(3H, singlet, --OCH₃), 4.96 tau (2H, doublet, --CH₂ OAc), 4.92 tau (1H,singlet, C₆ --H), 4.83 tau (1H, singlet, φ(N₃)C--H), 2.55 tau (5H,phenyl).

C. 7-(D-α-amino-2-phenylacetamido)-7-methoxycephalosporanic acid

To a solution of 620 mg. of7-(D-α-azido-2-phenylacetamido)-7-methoxycephalosporanic acid in 6.2 ml.of acetic acid and 9 ml. of water is added 3.1 g. of powdered zinc andthe solution stirred for 6 minutes at 0° C. The zinc is filtered off andwashed with 60 ml. of cold water. The combined filtrates are saturatedat 0° C. with hydrogen sulfide and through diatomaceous earth. Thefiltrate is washed with 3×50 ml. of ethylacetate and the aqueoussolution is warmed under reduced pressure to remove dissolvedethylacetate and finally lyophilized to afford 480 mg. of7-(D-α-amino-2-phenylacetamido)-7-methoxycephalosporanic acid as a whitepowder. This product contains 1 equivalent of acetic acid and 2equivalents of water and 2% ammonia as the acetate or antibiotic salt.Spinco analysis shows 1.58 micromoles/mg. phenylglycine (84% of theory).TGA finds 17.8% weight loss to 110° C. (99% of theory). Titration:inflections at pH 5.7 and 8.7, pH 1/2=7.0, EW 476 (theory for acetatedihydrate=515). Electrophoresis at pH 7 shows a single spot as amonoanion. Calculated for C₁₉ H₂₁ N₃ O₇.2H₂ O.AcOH+2% NH₃ : C, 46.2; H,5.7; N, 8.8; S, 5.6. Found: C, 47.41; H, 4.99; N, 9.48; S, 6.36.Distillation from alkali and titration of the distillate finds 2% NH₃.UV (pH 7 buffer): λ_(max).=263, E% 116 (ε=6170). The NMR spectrum (100MHz, D₂ O) has bands at 7.65 tau (AcOH, ca. 1 equivalent), 7.61 tau(singlet, --COCH₃), 6.17 tau (singlet, --OCH₃), 6.02, 6.19, 6.45, 6.62tau (AB qt., J=Hz, S--CH₂ --), 2.13 tau (singlet, phenyl); HOD at 5 tauobscures the other protons. IR spectrum (Nujol): 2.8-4.5μ (NH₃ ⁺), 5.65μ(β-lactam), 5.85μ (ester), 6.2-6.3μ (COO⁻).

EXAMPLE 14 A. Benzhydryl7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanate

To a solution of 0.5 g. of benzhydryl 7-amino-7-methoxycephalosporanatein 15 ml. of methylene chloride is added the monobenzhydrylphenylmalonyl chloride (prepared as described below) followed by 0.5 ml.of pyridine. The resulting reaction mixture is stirred for 30 minutesand then 12 ml. of water is added. The aqueous mixture is stirred for 5minutes and the layers separated. The methylene chloride portion iswashed with 2.5 N HCl, water, twice with aqueous sodium bicarbonate andsaturated sodium chloride. The solvent layer is then dried overmagnesium sulfate, filtered and evaporated below 25° C. under reducedpressure to afford 0.695 g. of benzhydryl7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanate. This ischromatographed on 50 g. of neutral silica gel and eluted withchloroform to afford 400 mg. of product as a tan glass. IR: 3.03μ (NH),5.62μ (β-lactam), 5.78μ (ester), 5.88μ (amide). NMR (CCl₄): 8.18 tau(3H, singlet, acetyl), 7.0 tau (2H, S--CH₂ --), 6.80, 6.73 tau (3H,doublet, diastereomeric OCH₃ 's), 5.2 tau (4H, multiplet, --CH₂ O--, C₆proton, malonyl CH), 3.17 tau (2H, CHφ₂), 2.8 tau (20H, aromatic), 2.0tau (1H, amide NH).

The monobenzhydryl phenylmalonyl chloride used above is prepared asfollows: To a solution of 19.25 g. of phenylmalonic acid in 165 ml. ofethylacetate is added a solution of 25 g. of diphenyldiazomethane in 100ml. of ethylacetate over 15 minutes at 15°-20° C. The solution isstirred 10 minutes more and 500 ml. of water is added, and sufficient50% sodium hydroxide is added at 15° C. to make the reaction mixturealkaline. The solvent layer of the mixture is separated, extracted twicewith aqueous sodium bicarbonate solution. The combined aqueous solutionsare washed twice with ethylacetate, cooled, acidified with hydrochloricacid and extracted 3 times with ethylacetate. The ethylacetateextractions are washed twice with water, once with saturated sodiumchloride solution and then dried over magnesium sulfate. The solvent isthen evaporated at a temperature below 25° C. under reduced pressure toan oil which is crystallized from 200 ml. of 1:3 ether in petroleumether to afford 20.9 g. of monobenzhydryl phenylmalonate, m.p.119.5°-122° C. To a slurry of 0.7 g. of this monoester in 2.5 ml. ofwater is added 2.10 ml. of 0.962 N sodium hydroxide. The solution isstirred for 3 minutes, filtered and freeze dried to obtain the sodiumsalt of the monoester. To this sodium salt is added 5 ml. of benzene andthe slurry is treated at 0° C. with 1.5 ml. of oxalyl chloride. After 10minutes at 0° C. and 5 minutes at 25° C. the mixture is evaporated at atemperature below 25° C. under reduced pressure and the residue is twiceconcentrated from carbon tetrachloride. Under a dry atmosphere, theproduct in 5 ml. of carbon tetrachloride is filtered and concentrated toafford the monobenzhydryl phenylmalonyl chloride.

B. Disodium 7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanate

Benzhydryl 7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanate(400 mg.) is treated at 0° C. for 2 minutes with 1.2 ml. of anisole and6 ml. of trifluoroacetic acid. The resulting reaction product is thenfrozen, stripped at low temperature and high vacuum, diluted withanisole and stripped once again at 25° C. The residue comprising thefree acid is taken up in 20 ml. of 1 molar sodium bicarbonate solution,washed 4 times with small amounts of methylene chloride, acidified withHCl, saturated with NaCl and extracted with 4×10 ml. of ethylacetate.The solvent extractions are washed twice with saturated sodium chloride,dried with magnesium sulfate, filtered and evaporated under reducedpressure to afford 218 mg. of7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanic acid as ayellow syrup. This is dissolved in 5 ml. of water containing 79 mg. ofsodium bicarbonate and freeze dried. The freeze dried residue isdissolved in 5 ml. of water, filtered, and lyophilized again to afford182 mg. of disodium7-(2-carboxy-2-phenylacetamido)-7-methoxycephalosporanate. The productso obtained is combined with 550 mg. of the same product obtained from asecond run and dissolved in 10 ml. of water. The resulting solution isfiltered and lyophilized to give 647 mg. of product. UV (pH 7): λmax.265 nm., ε=6300. NMR (D₂ O): 7.90 tau (3H, singlet, acetyl), 6.70, 6.54tau (2H, 2 AB quartets, diastereomeric S--CH₂ --), 6.50, 6.38 tau (3H,2singlets, diastereomeric OCH₃ 's), 5.22 tau (2H, broad s, --CH₂ O--),4.85 tau (1H, singlet, C₆ proton), 2.55 tau (5H, singlet, aromatic). Thelarge HOD peaks covers the malonyl proton adsorption. On electrophoresisat pH 7, the product moves as a dianion, single spot. The pH of a 10%aqueous solution is 8.8. Calculated for C₂₀ H₁₈ N₂ SO₄ Na₂ +Na₂ CO₃ +0.4NaHCO₃ +H₂ O: C, 38.59; H, 3.09; N, 4.21; S, 4.81; ash (as Na) 15.19.Found: C, 38.99; H, 3.10; N, 4.17; S, 4.91; ash (as Na) 15.3.

EXAMPLE 15 A. Benzhydryl 7-azido-7-benzyloxycephalosporanate

A mixture of 2.4 g. of benzhydryl 7-azido-7-bromocephalosporanateprepared as described in Example 2C and 1.5 g. of silvertetrafluoroborate in 10 ml. of benzyl alcohol is stirred at roomtemperature for 3 hours. The mixture is diluted with 300 ml. of etherand the silver salts removed by filtration. The filtrate is washed withaqueous sodium bicarbonate, dried over sodium sulfate and evaporatedunder reduced pressure. The excess benzyl alcohol is removed on a highvacuum pump with magnetic stirring for 18 hours and the residuechromatographed on silica gel. Elution with hexane followed byincreasing concentrations of methylene chloride in hexane andevaporation of the fractions containing the product affords benzhydryl7-azido-7-benzyloxycephalosporanate.

B. Benzhydryl 7-benzyloxy-7-phenylacetamidocephalosporanate

A solution of 1.2 g. of the benzhydryl7-azido-7-benzyloxycephalosporanate in 25 ml. of dry ethylacetate ishydrogenated in the presence of 1.2 g. of 10% palladium-on-charcoalcatalyst for 18 hours at room temperature. The catalyst is removed byfiltration and phenylacetic anhydride (1.0 g.) is added to the filteredsolution and the mixture kept for 45 minutes at room temperature. Theresulting reaction mixture is diluted with 200 ml. of ether, 200 ml. ofpH 7 phosphate buffer is added, and the mixture stirred vigorously for1/2 hour. The ether layer is separated, washed with water, dried andevaporated under reduced pressure. The residue is chromatographed on 50g. of silica gel and eluted with 5% ethylacetate in methylene chloridewhich, on evaporation, affords benzhydryl7-benzyloxy-7-phenylacetamidocephalosporanate.

C. Sodium 7-benzyloxy-7-phenylacetamidocephalosporanate

A solution of 0.5 g. of benzhydryl7-benzyloxy-7-phenylacetamidocephalosporanate in 3.5 ml. of anisole istreated with 10 ml. of trifluoroacetic acid at room temperature for 10minutes. The trifluoroacetic acid and anisole are removed under reducedpressure at a temperature below 40° C. and the residue is taken up in 25ml. of chloroform and treated with 20 ml. of water containing 0.15 g. ofsodium bicarbonate. The mixture is stirred for 1/2 hour at roomtemperature and the organic phase is separated and washed with water.The combined aqueous phase is washed twice with methylene chloride andthe water layer and washes combined and lyophilized affording sodium7-benzyloxy-7-phenylacetamidocephalosporanate.

EXAMPLE 16 A. Benzhydryl 7-azido-7-methylmercaptocephalosporanate

To a solution of 2 g. of benzhydryl 7-diazocephalosporanate prepared asdescribed in Example 2B in 100 ml. of methylene chloride at -75° C.under an atmosphere of nitrogen is added a solution of 1 ml. ofmethylsulfenyl chloride in 10 ml. of methylene chloride with vigorousstirring. Nitrogen is immediately evolved. After the addition of thereagent over a period of about 10 minutes, the mixture is brought to -5°C. gradually in about 1/2 hour. Saturated sodium bicarbonate is added anthe organic layer is separated and washed with water. After drying oversodium sulfate the solvent is removed at room temperature under reducedpressure affording benzhydryl 7-chloro-7-methylmercaptocephalosporanate.This product is stirred with a 20 ml. solution of trimethylammoniumazidein methylene chloride at 0°-5° C. under nitrogen for 11/2 hours. Thesolution is then washed with cold saturated sodium bicarbonate solution,water, and dried over sodium sulfate. Evaporation of the solvent affordsthe crude benzhydryl 7-azido-7-methylmercaptocephalosporanate.

B. Benzhydryl 7-methylmercapto-7-phenylacetamidocephalosporanate

A solution of 1.2 g. of the benzhydryl7-azido-7-methylmercaptocephalosporanate in 25 ml. of dry ethylacetatecontaining 0.8 ml. of diisopropylethylamine is hydrogenated in thepresence of 1.2 g. of 10% palladium-on-charcoal catalyst for 18 hours atroom temperature. Phenylacetic anhydride (0.1 g.) is added and themixture kept for 45 minutes at room temperature. The mixture is dilutedwith 200 ml. of ether, 200 ml. of pH 7 phosphate buffer is added, andthe mixture stirred vigorously for 1/2 hour. The ether layer isseparated, washed with water, dried and evaporated to afford benzhydryl7-methylmercapto-7-phenylacetamidocephalosporanate

C. Sodium 7-methylmercapto-7-phenylacetamidocephalosporanate

A solution of 0.4 g. of benzhydryl7-methylmercapto-7-phenylacetamidocephalosporanate in 3.5 ml. of anisoleis treated with 10 ml. of trifluoroacetic acid at room temperature for10 minutes. The trifluoroacetic acid and anisole are removed underreduced pressure, maintaining the temperature below 40° C., and theresidue is taken up in 25 ml. of chloroform and treated with 20 ml. ofwater containing 0.12 g. of sodium bicarbonate. The mixture is stirredfor 1/2 hour at room temperature and the organic phase is separated andwashed with water. The combined aqueous phase is washed twice withmethylene chloride and then lyophilized to give sodium7-methylmercapto-7-phenylacetamidocephalosporanate.

EXAMPLE 17 A. Benzhydryl 7-azido-7-ethoxycephalosporanate

To a solution of 217 mg. benzhydryl 7-azido-7-bromocephalosporanate in15 ml. of absolute ethanol is added 31.6 μl. of pyridine and 78 mg. ofsilver fluoroborate and the mixture stirred at room temperature for 2hours, protected from light and moisture. The mixture is evaporated todryness in vacuo and the residue chromatographed on 20 g. of silica gel.Elution with chloroform affords 121 mg. of benzhydryl7-azido-7-ethoxycephalosporanate, m.p. 144.5°-145° C.

B. Benzhydryl 7-ethoxy-7-thienylacetamidocephalosporanate

A solution of 320 mg. of benzhydryl 7-azido-7-ethoxycephalosporanate in30 ml. of dioxane is stirred with 320 mg. of platinum oxide at roomtemperature under an atmosphere of hydrogen for 1 hour. An additional320 mg. of catalyst is introduced and the hydrogenation continued for 5hours. The mixture is evaporated under vacuum to dryness and the residuetaken up in chloroform and filtered through 2 inches of silica gel andevaporated, leaving benzhydryl 7-amino-7-ethoxycephalosporanate as ayellow oil. This is dissolved in methylene chloride and to the cooledsolution is added with stirring 0.2 ml. of thienylacetyl chloride and0.2 ml. of dry pyridine. The mixture is stirred for 15 minutes at 0° C.and then poured over ice and the methylene chloride layer separated,washed with aqueous sodium bicarbonate solution, dried and evaporated.The residue is chromatographed on 20 g. of silica gel. Elution withchloroform affords 120 mg. of benzhydryl7-ethoxy-7-thienylacetamidocephalosporanate. TLC 2% MeOH in CH₂ Cl₂ Rf0.73. IR: 5.65μ (lactam); 5.85, 5.95; 6.0 (amide). NMR: 8.82 tau (CH₂CH₃), ##STR61## 6.64-6.67 tau (doublet, SCH₂), 6.14 tau (singlet,thienyl CH₂), 4.96 tau (singlet, 6H).

C. Sodium 7-ethoxy-7-thienylacetamidocephalosporanate

A solution of 0.9 g. of benzhydryl7-ethoxy-7-thienylacetamidocephalosporanate in 6.35 ml. anisole and 17.1ml. of trifluoroacetic acid is kept at room temperature for 5 minutesand then evaporated under high vacuum. The residue is stirred with waterand methylene chloride and the pH adjusted to 5.8 by the addition ofsodium bicarbonate solution. The aqueous layer is evaporated andacidified to pH 2 and the gummy precipitate filtered off. This isdissolved in butylacetate, filtered and the filtrate stirred with waterwith the addition of sodium bicarbonate solution to pH 6.5. The aqueousextract is evaporated and freezedried, leaving 0.2 g. of sodium7-ethoxy-7-thienylacetamidocephalosporanate. Circular paperchromatography in butanolethanol-water (4:1:5) gives a spot of Rf 0.55.Analysis calculated: C, 46.74; H, 4.14; N, 6.00. Found: C, 47.73; H,4.09; N, 5.73.

EXAMPLE 18 A. Benzhydryl7-(dl-α-fluorophenylacetamido)-7-methoxycephalosporanate

To a solution of 0.9 g. of benzhydryl 7-amino-7-methoxycephalosporanatein 45 ml. of methylene chloride at 0° C. is added with stirring 0.96 ml.of dl-α-fluorophenylacetyl chloride. After 1 minute 0.96 ml. of pyridineis added and the mixture stirred for 15 minutes. The reaction mixture isthen poured over ice, shaken and the methylene chloride layer isremoved. The aqueous layer is extracted twice with methylene chloride.The combined aqueous layers are washed with aqueous sodium bicarbonate,then with water and dried over sodium. The solvent is evaporated toafford 1.2 g. of benzhydryl7-(dl-α-fluorophenylacetamido)-7-methoxycephalosporanate. The product inmethylene chloride solution is chromatographed over 90 g. of silica gel.The silica gel column is developed successively with 1,000 ml. ofmethylenechloride, 500 ml. of methylene chloride containing 5%chloroform, and then 300 ml. each of ethylene chloride containing 10%,25% and 50% chloroform, respectively. The pooled fractions of the 50%chloroform eluates are evaporated under reduced pressure to afford 370mg. of product. Analysis calculated: C, 63.56; H, 4.83; N, 4.63. Found:C, 62.71; H, 5.02; N, 4.38. TLC in 5% ethylacetate in methylene chlorideshows two spots at Rf 0.406 and 0.54.

B. Sodium 7-(dl-α-fluorophenylacetamido)-7-methoxycephalosporanate

To a solution of 360 mg. of benzhydryl7-(dl-α-fluorophenylacetamido)-7-methoxycephalosporanate in 2.44 ml. ofanisole is added 6.9 ml. of trifluoroacetic acid and the reactionallowed to proceed for 15 minutes. The mixture is evaporated underreduced pressure and the residue taken up in a mixture of water andmethylene chloride. The pH of the mixture is adjusted to 6.8 and theaqueous layer freeze-dried to afford sodium7-(dl-α-fluorophenylacetamido)-7-methoxycephalosporanate, Rf inbutanol-ethanol-water (4:1:5) 0.41 and 0.60.

EXAMPLE 19 A. 3-Methyl-7β-trichloroethoxycarboxamidodecephalosporanicacid

To a solution of 21.4 g. of 3-methyl-7-aminodecephalosporanic acid in250 ml. of water adjusted to pH 8.7 with 15% sodium hydroxide solutionis added 21.2 g. of trichloroethoxycarbonyl chloride over a half hourperiod while keeping the temperature at 20° C. and maintaining the pH at8.7 by means of a pH stat. The reaction mixture is allowed to stir foranother half hour at this temperature and then adjusted to pH 2 andextracted into ethylacetate using three extractions. The ethylacetatelayer is washed twice with water, dried over anhydrous sodium sulfate,and evaporated under reduced pressure to give3-methyl-7β-trichloroethoxycarboxamidodecephalosporanic acid.

The 3-methyl-7-aminodecephalosporanic acid used as the starting materialin this example is produced by the catalytic reduction of cephalosporinC followed by hydrolytic removal of the 5-amino adipoyl side chain asdescribed in U.S. Pat. No. 3,129,224.

B. p-Methoxybenzyl3-methyl-7β-trichloroethoxycarboxamidodecephalosporanate

3-Methyl-7β-trichloroethoxycarboxamidodecephalosporanic acid (22.38 g.)and triethylamine (14 ml.) are stirred in 200 ml. of acetone for 45minutes and p-methoxybenzyl bromide (10.1 g.) is added to this mixtureand stirring continued for an additional 20 minutes at room temperature.The entire reaction mixture is then chilled in an ice bath and theprecipitated triethylamine hydrobromide is removed by filtration. Thefiltrate is evaporated under reduced pressure and the residue isextracted by shaking with a mixture of ether and aqueous sodium hydrogenphosphate. The ethereal layer is dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give p-methoxybenzyl3-methyl-7β-trichloroethoxycarboxamidodecephalosporanate.

C. p-Methoxybenzyl 3-methyl-7-aminodecephalosporanate

The trichloroethoxycarbonyl protecting group is removed by adding anethylacetate solution (50 ml.) containing p-methoxybenzyl3-methyl-7β-trichloroethoxycarboxamidodecephalosporanate (10 g.) to avigorously stirred slurry of 5 g. of zinc dust in 50 ml. of 90% aceticacid in an ice water bath over 5-10 minutes. The temperature is kept at25°-30° C. After 2 hours the solids are removed by filtration and washedwith 100 ml. of ethylacetate. The combined filtrate is diluted with 200ml. water, the layers separated and the organic layer washed with 100ml. water. The organic layers are washed with cold sodium bicarbonatesolution, dried and concentrated under reduced pressure. The residue ischromatographed on silica gel (40 g.) using CHCl₃ :MeOH mixtures aseluants to give p-methoxybenzyl 3-methyl-7-aminodecephalosporanate.

D. p-Methoxybenzyl 3-methyl-7-diazodecephalosporanate

To a stirring mixture of 1.6 g. of sodium nitrite, 30 ml. of water and40 ml. of methylene chloride at 0° C. is added 0.8 g. of p-methoxybenzyl3-methyl-7-aminodecephalosporanate followed by the addition of asolution of 0.75 g. of p-toluenesulfonic acid in 5 ml. of water over afew minutes. The mixture is stirred at 0° C. for 20 minutes and theorganic phase is separated, washed with ice water, dried over anhydroussodium sulfate, filtered and concentrated under reduced pressure at roomtemperature to afford p-methoxybenzyl3-methyl-7-diazodecephalosporanate.

E. Sodium 3-methyl-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Following the procedures described in Examples 2C, 2D, 3A, 3B and 3C,p-methoxybenzyl 3-methyl-7-diazodecephalosporanate is converted tosodium 3-methyl-7-methoxy-7-(2-thienylacetamido)decephalosporanate.

EXAMPLE 20 A. o-Nitrobenzyl3-methyl-7β-trichloroethoxycarboxamidodecephalosporanate

When p-methoxybenzyl bromide in Example 19B is replaced by an equivalentamount of o-nitrobenzyl bromide and the resulting reaction product isrecovered using the described processes, the corresponding o-nitrobenzylester is obtained.

D. o-Nitrobenzyl 3-methyl-7-aminodecephalosporanate

The trichloroethoxycarbonyl protecting group is removed and the productis recovered using the procedures described in Example 19C to affordo-nitrobenzyl 3-methyl-7-aminodecephalosporanate.

C. o-Nitrobenzyl 3-methyl-7-diazodecephalosporanate

Diazotization of the product of B above and recovery of the diazoproduct is carried out using the procedures described in Example 19D.

D. o-Nitrobenzyl 3-methyl-7-azido-7-bromodecephalosporanate

When o-nitrobenzyl 3-methyl-7-diazodecephalosporanate is reacted withtriethylammonium azide and bromine azide, the resulting reaction productis recovered in accordance with the procedures described in Example 2Cto afford o-nitrobenzyl 3-methyl-7-azido-7-bromodecephalosporanate.

E. o-Nitrobenzyl 3-methyl-7-azido-7-methoxydecephalosporanate

The product of D above is reacted with methanol in the presence ofsilver fluoroborate and the resulting reaction product is recovered asdescribed in Example 1C to obtain the title compound.

F. o-Nitrobenzyl3-methyl-7-(2-furylacetamido)-7-methoxydecephalosporanate

o-Nitrobenzyl 3-methyl-7-azido-7-methoxydecephalosporanate ishydrogenated and the resulting 7-amino compound is reacted withfurylacetyl chloride and is recovered in accordance with proceduresdescribed in Example 8 to obtain o-nitrobenzyl3-methyl-7-(2-furylacetamido)-7-methoxydecephalosporanate.

G. Sodium 3-methyl-7-(2-furylacetamido)-7-methoxydecephalosporanate

A 0.5% solution of o-nitrobenzyl3-methyl-7-(2-furylacetamido)-7-methoxydecephalosporanate in dioxane isplaced in a pyrex reaction vessel, thoroughly de-gassed and purged withhelium. The solution is then irradiated with 3000 nM light until theester group is removed as evidenced by TLC. After evaporating thesolvent under reduced pressure, the residue is taken up in 15 ml. ofmethylene chloride and stirred for 1/2 hour with a solution of oneequivalent of sodium bicarbonate in 10 ml. of water. The aqueous layeris lyophilized to give a mixture containing sodium3-methyl-7-(2-furylacetamido)-7-methoxydecephalosporanate.

EXAMPLE 21 A.3-Benzoylthiomethyl-7β-t-butoxycarboxamidodecephalosporanic acid

A solution of 35 g. of 3-benzoylthiomethyl-7-aminodecephalosporanic acidin 250 ml. of water is adjusted to pH 8.7 with 15% sodium hydroxidesolution and maintained at this pH throughout the reaction by means of apH stat. To this solution at 20° C. is added t-butoxychloroformate overa half hour period. After continuing the stirring for another half hour,the reaction mixture is adjusted to pH 2 and extracted into ethylacetateusing three extractions. The ethylacetate solution is washed twice withwater and dried over anhydrous sodium sulfate. After filtration thesolvent is removed under reduced pressure to afford3-benzoylthiomethyl-7β-t-butoxycarboxamidodecephalosporanic acid.

The starting compound is a known derivative of 7-aminocephalosporanicacid described in Belgian Pat. No. 650,444.

B. Phenacyl 3-benzoylthiomethyl-7β-t-butoxycarboxamidodecephalosporanate

3-Benzoylthiomethyl-7β-t-butoxycarboxamidodecephalosporanic acid (35.0g.) and triethylamine (23 ml.) are stirred in 200 ml. of acetone for 45minutes and 15.9 g. of α-bromoacetophenone is added to the mixture andstirring continued for an additional 20 minutes at room temperature. Theentire reaction mixture is then chilled in an ice bath and thetriethylamine hydrobromide is removed by filtration. The filtrate isevaporated under reduced pressure and the residue is extracted byshaking with a mixture of ether and aqueous sodium hydrogen phosphate.The ethereal layer is dried over anhydrous sodium sulfate andconcentrated under reduced pressure to give phenacyl3-benzoylthiomethyl-7β-butoxycarboxamidodecephalosporanate.

C. Phenacyl 3-benzoylthiomethyl-7-aminodecephalosporanate

Phenacyl 3-benzoylthiomethyl-7β-t-butoxycarboxamidodecephalosporanate(10 g.) is dissolved in 35 ml. of anisole and 100 ml. of trifluoroaceticacid at room temperature for 10 minutes. The solvents are removed underreduced pressure, maintaining the temperature below 40° C. The residueis taken up in water and neutralized with solid sodium bicarbonate.Chloroform is added and the chloroform layer is separated, washed withwater and dried. The organic extract is concentrated under reducedpressure to give phenacyl 3-benzoylthiomethyl-7-aminodecephalosporanate.

D. Phenacyl 3-benzoylthiomethyl-7-diazodecephalosporanate

A mixture of 10 g. of sodium nitrite, 4.5 g. of phenacyl3-benzoylthiomethyl-7-aminodecephalosporanate, 300 ml. of methylenechloride and 300 ml. of a mixture of water and ice is shaken in aseparatory funnel. p-Toluenesulfonic acid monohydrate (0.27 g.) is addedin three portions during 20 minutes. The mixture is shaken vigorouslyand the methylene chloride layer is separated, dried over sodium sulfateand evaporated under reduced pressure to 40 ml. The solution of phenacyl3-benzoylthiomethyl-7-diazodecephalosporanate is used in the next stepwithout isolation of the intermediate product.

E. Sodium3-benzoylthiomethyl-7-(2-furylacetamido)-7-methoxydecephalosporanate

Following the procedures described in Example 1B and 1C using phenacyl3-benzoylthiomethyl-7-diazodecephalosporanate in place of benzyl7-diazodecephalosporanate, the diazo compound is converted to phenacyl3-benzoylthiomethyl-7-azido-7-methoxydecephalosporanate. The latterproduct is then converted to sodium3-benzoylthiomethyl-7-(2-furylacetamido)-7-methoxydecephalosporanatefollowing the procedures described in Example 8.

EXAMPLE 22 A. Trimethylsilyl3-carbamoyloxymethyl-7-aminodecephalosporanate

A mixture of 0.5 g. of 3-carbamoyloxymethyl-7-aminodecephalosporanicacid, 2 ml. of hexamethyldisilazane and 8 ml. of chloroform is stirredovernight at reflux temperature protected from moisture. The solvent andexcess hexamethyldisilazane are removed at reduced pressure, leaving aresidue containing trimethylsilyl3-carbamoyloxymethyl-7-aminodecephalosporanate.

B. Trimethylsilyl 3-carbamoyloxymethyl-7-diazodecephalosporanate

A mixture of trimethylsilyl3-carbamoyloxymethyl-7-aminodecephalosporanate, 0.05 ml. oftrifluoroacetic acid and 5 ml. of chloroform is stirred at 0° C. and 0.2ml. of isoamylnitrite is added. After stirring for 1 hour at 0° C. theresulting reaction mixture containing the trimethylsilyl3-carbamoyloxymethyl-7-diazodecephalosporanate is used directly in thenext step.

C. 3-Carbamoyloxymethyl-7-azido-7-bromodecephalosporanic acid

To the solution obtained in B above is added at 0° C. 5 ml. ofnitromethane. This is followed in rapid succession by 1 ml. of amethylene chloride solution of trimethylammonium azide and 1.5 ml. of amethylene chloride solution of bromine azide, both of which are inconsiderable excess of the amount required. The bromine size isdecolorized rapidly and nitrogen is evolved. After about 5-10 minutes 20ml. of 0.1 N sodium thiosulfate is added and the layers are separated.The aqueous phase is adjusted to pH 2 by the addition of a few drops ofhydrochloric acid and is extracted twice with methylene chloride. Thecombined extractions are washed with water, dried over anhydrous sodiumsulfate and concentrated under reduced pressure to afford3-carbamoyloxymethyl-7-azido-7-bromodecephalosporanic acid.

The 3-carbamoyloxymethyl-7-aminodecephalosporanic acid used as thestarting material in this example can be prepared by the followingprocedures:

7-Aminocephalosporanic acid is reacted with t-butoxycarbonylazide toproduce the 7β-(t-butoxycarbonyl) derivative in accordance with knownmethods. This derivative is then intimately contacted with citrusacetylesterase in aqueous phosphate buffer at pH 6.5-7 for 15 hours and3-hydroxymethyl 7β-(t-butoxycarbonyl)aminodecephalosporanic acid isrecovered from the resulting reaction mixture.

To 0.2 g. of 3-hydroxymethyl 7β-(t-butoxycarbonyl)aminodecephalosporanicacid suspended in 5 ml. of acetonitrile, cooled to 0° C. and maintainedunder nitrogen atmosphere is added 0.15 ml. of chlorosulfonylisocyanate. The reaction mixture is stirred for 70 minutes and thenevaporated under diminished pressure to dryness. The resulting residueis taken up in 10 ml. of ethylacetate and 10 ml. of 0.1 N phosphatebuffer. The pH of the aqueous layer is adjusted to about 1.6 and themixture stirred for 21/2 hours at room temperature. The pH is thenadjusted to about 8 with aqueous tripotassium phosphate solution, andthe aqueous phase is separated. The organic phase is reextracted with 10ml. of phosphate buffer at pH 8. The combined aqueous phase is adjustedto pH 2.1 with hydrochloric acid and extracted twice with ethylacetate.The ethylacetate extractions are dried over sodium sulfate andevaporated under diminished pressure to afford 0.055 g. of residue. Thisresidue is washed with ether to afford3-carbamoyloxymethyl-7β-(t-butoxycarbonyl)aminodecephalosporanic acidwhich is recovered as a yellow solid.

3-Carbamoyloxymethyl-7β-(t-butoxycarbonyl)aminodecephalosporanic acid(0.5 g.) in 3.5 ml. of anisole is stirred with 2 ml. of trifluoroaceticacid at 0° C. for 5 minutes. The resulting reaction mixture isevaporated under reduced pressure to afford3-carbamoyloxymethyl-7-aminodecephalosporanic acid which is purifiedfurther by crystallization from aqueous isopropanol.

EXAMPLE 23 Benzyl 3-picolinoylthiomethyl-7-diazodecephalosporanate

Following the procedures described in Example 1A, benzyl3-picolinoylthiomethyl-7-aminodecephalosporanate, obtained by theesterification of the free acid with benzyl chloride, is converted tothe corresponding diazo compound, benzyl3-picolinoylthiomethyl-7-diazodecephalosporanate.

The 3-picolinoylthiomethyl-7-aminodecephalosporanic acid is obtained byreacting 3-hydroxymethyl-7-aminodedephalosporanic acid withthiopicolinic acid following procedures described in British PatentSpecification No. 1,101,423.

EXAMPLE 24 Benzhydryl 3-pyridiniummethyl-7-diazodecephalosporanate

Following the procedures described in Example 2A and 2B,3-pyridiniummethyl-7-aminodecephalosporanic acid is converted to thebenzhydryl ester which, on reaction with nitrite, is converted to thedesired benzhydryl 3-pyridiniummethyl-7-diazodecephalosporanate.

The starting compound is prepared by treating cephalosporin C withpyridine followed by acid hydrolysis as described in U.S. Pat. No.3,117,126.

EXAMPLE 25 Benzhydryl3-N-(2-chlorethyl)carbamoyloxymethyl-7-diazodecephalosporanate

Following the procedures described in Example 2A,3-N-(2-chloroethyl)carbamoyloxymethyl-7-aminodecephalosporanic acid isconverted to the benzhydryl ester which is then diazotized following theprocedures described in Example 2B to obtain the desired7-diazodecephalosporanate.

The substituted aminodecephalosporanic acid used as the startingmaterial in this example can be prepared by subjecting the7-t-butyloxycarboxy-7-ACA to enzymatic hydrolysis to obtain thedesacetyl compound which is reacted with β-chloroethyl-isocyanate indimethylformamide to afford the3-N-(2-chloroethyl)carbamoyloxymethyldecephalosporin which is hydrolyzedto obtain 3-N-(2-chlorethyl)carbamoyloxymethyl-7-aminodecephalosporanicacid.

EXAMPLE 26 3-Hydroxymethyl-7-diazodecephalosporanic acid lactone

A mixture of 10 g. of sodium nitrite, 4 g. of3-hydroxymethyl-7-aminodecephalosporanic acid lactone, 300 ml. ofmethylene chloride and 300 ml. of water and ice is shaken in aseparatory funnel. p-Toluenesulfonic acid monohydrate (1.6 g.) is addedin 3 portions during 20 minutes. The reaction mixture is shakenvigorously during the addition of the sulfonic acid. The methylenechloride layer is separated, dried over sodium sulfate and evaporatedunder reduced pressure to afford the desired3-hydroxymethyl-7-diazodecephalosporanic acid lactone.

The 3-hydroxymethyl-7-aminodecephalosporanic acid lactone is obtained byacid hydrolysis of cephalosporin C in accordance with procedures knownin this art.

EXAMPLE 27 t-Butyl-7-diazocephalosporanate

To a stirring mixture of 1.6 g. of sodium nitrite, 30 ml. of water and40 ml. of methylene chloride at 0° C. is added 0.8 g. oft-butyl-7-aminocephalosporanate followed by the addition of a solutionof 0.8 g. of p-toluenesulfonic acid in 5 ml. of water; the acid beingadded in small portions over a period of about 10 minutes. The resultingmixture is stirred at 0° C. for 20 minutes and the organic phase is thenseparated, washed with 1×10 ml. of ice water, dried over anhydroussodium sulfate at 0° C., filtered and concentrated under reducedpressure at room temperature to afford thet-butyl-7-diazocephalosporanate.

EXAMPLE 28 Benzhydryl 3-n-amyloxymethyl-7-diazodecephalosporanate

To a slurry of 6.8 g. of 3-n-amyloxymethyl-7-aminodecephalosporanic acidin 300 ml. of dioxane at room temperature is added with stirring 4.3 g.of p-toluenesulfonic acid monohydrate. The resulting solution isconcentrated under reduced pressure and flushed twice with dioxane. Theresidue is dissolved in 300 ml. of dioxane at room temperature, and asolution of 10 g. of diphenyldiazomethane in 25 ml. of dioxane is addeddropwise over 15 minutes. The solution is stirred for an additional 30minutes and then 25 ml. of methanol is added to destroy the excessdiphenyldiazomethane. The mixture is concentrated under reduced pressureand the residue partitioned between 200 ml. of methylene chloride and200 ml. of water containing 10 g. of dipotassium sulfate (pH 8.5). Theorganic phase is washed with water, dried over sodium sulfate andconcentrated under reduced pressure to afford the desired benzhydryl3-n-amyloxymethyl-7-aminodecephalosporanate.

To a stirring mixture of 1.6 g. of sodium nitrite, 30 ml. of water and40 ml. of methylene chloride at 0° C. is added 0.8 g. of the esterprepared above followed by the addition of a solution of 0.8 g. ofp-toluenesulfonic acid in 5 ml. of water over a few minutes. The mixtureis stirred at 0° C. for 20 minutes and the organic phase is separated,washed with a small amount of ice water, dried over anhydrous sodiumsulfate at 0° C., filtered and concentrated under reduced pressure atroom temperature to afford the benzhydryl3-n-amyloxymethyl-7-diazodecephalosporanate.

EXAMPLES 29-32

The p-methoxybenzyl 3-methyl-7-diazodecephalosporanate obtained inExample 19D, the phenacyl 3-benzoylthiomethyl-7-diazodecephalosporanate,prepared as in Example 21D, the benzyl3-picolinoylthiomethyl-7-diazodecephalosporanate of Example 23, thebenzhydryl 3-pyridiniummethyl-7-diazodecephalosporanate of Example 24,the benzhydryl3-N-(2-chloroethyl)carbamoyloxymethyl-7-diazodecephalosporanate ofExample 25, and the benzhydryl3-n-amyloxymethyl-7-diazodecephalosporanate of Example 28 are eachreacted with trimethylammonium azide and bromine azide, and theresulting reaction product is isolated following the procedure describedin Example 2C to afford the following products:

    ______________________________________                                        Example No. Product                                                           ______________________________________                                        29          p-Methoxybenzyl 3-methyl-7-azido-7-bromo-                                     decephalosporanate                                                30          Phenacyl 3-benzoylthiomethyl-7-azido-7-                                       bromodecephalosporanate                                           31          Benzyl 3-picolinoylthiomethyl-7-azido-7-                                      bromodecephalosporanate                                           32          Benzhydryl 3-pyridiniummethyl-7-azido-7-                                      bromodecephalosporanate                                           ______________________________________                                    

EXAMPLE 33 A. p-Methoxybenzyl3-methyl-7-azido-7-chlorodecephalosporanate

p-Methoxybenzyl 3-methyl-7-diazodecephalosporanate (0.9 g.) is dissolvedin a mixture of 10 ml. of methylene chloride and 10 ml. of nitromethane.To this cooled solution at 0°-10° C. is added a solution oftriethylammonium azide prepared as described in Example 2C and amethylene chloride solution of chlorineazide (0.31 N, 15 ml.) and then50 ml. of water. The resulting reaction mixture is adjusted to pH 8 bythe addition of solid sodium bicarbonate. The mixture is allowed tostand and the organic layer is separated, extracted with 2×20 ml. ofwater, dried over anhydrous sodium sulfate and concentrated underreduced pressure to afford p-methoxybenzyl3-methyl-7-azido-7-chlorodecephalosporanate.

B. When an equivalent amount of benzyl 7-diazocephalosporanate,benzhydryl 7-diazocephalosporanate, phenacyl3-benzoylthiomethyl-7-diazodecephalosporanate, trimethylsilyl3-carbamoyloxymethyl-7-diazodecephalosporanate, benzyl3-picolinoylthiomethyl-7-diazodecephalosporanate, benzhydryl3-pyridiniummethyl-7-diazodecephalosporanate, benzhydryl3-N-(2-chloroethyl)carbamoyloxymethyl-7-diazodecephalosporanate,t-butyl-7-diazocephalosporanate or benzhydryl3-n-amyloxymethyl-7-diazodecephalosporanate is substituted for thep-methoxybenzyl 3-methyl-7-diazodecephalosporanate in the process ofExample 33A, the following compounds are obtained:

Benzyl7-azido-7-chlorocephalosporanate

Benzhydryl 7-azido-7-chlorocephalosporanate

Phenacyl 3-benzoylthiomethyl-7-azido-7-chlorodecephalosporanate

Trimethylsilyl 3-carbamoyloxymethyl-7-azido-7-chlorodecephalosporanate

Benzyl 3-picolinoylthiomethyl-7-azido-7-chlorodecephalosporanate

Benzhydryl 3-pyridiniummethyl-7-azido-7-chlorodecephalosporanate

Benzhydryl3-N-(2-chloroethyl)carbamoyloxymethyl-7-azido-7-chlorodecephalosporanate

t-Butyl-7-azido-7-chlorocephalosporanate

Benzhydryl 3-n-amyloxymethyl-7-azido-7-chlorodecephalosporanate.

EXAMPLE 34 A. Benzhydryl 7-azido-7-(2-bromoethoxy)cephalosporanate

A solution of 543 mg. benzhydryl 7-azido-7-bromocephalosporanate in 25ml. dry acetonitrile is treated with 195 mg. silver fluoroborate and 150mg. 2-bromoethanol. The mixture is stirred for 24 hours at roomtemperature, filtered, and evaporated at 30° C./0.1 mm. The residue ischromatographed on silica gel, eluting with chloroform to afford purebenzhydryl 7-azido-7-(2-bromoethoxy)cephalosporanate.

B. Benzhydryl 7-(2-thienylacetamido)-7-(2-bromoethoxy)cephalosporanate

Benzhydryl 7-azido-7-(2-bromoethoxy)cephalosporanate (587 mg.) ishydrogenated at 40 psi for 3 hours using 1 g. platinum oxide in 50 ml.dioxane containing 500 mg. thienylacetic anhydride. After filtration ofthe catalyst, the solution is treated for 15 minutes with 0.5 ml. waterto hydrolyze excess anhydride, then vacuum stripped. The crude productis taken up in 25 ml. ethylacetate, washed with aqueous sodiumbicarbonate, dried over magnesium sulfate, filtered, evaporated, andchromatographed on silica gel using 4:1 chloroform-ethylacetate to elutepure benzhydryl7-(2-thienylacetamido)-7-(2-bromoethoxy)cephalosporanate.

C. Benzhydryl7-(2-thienylacetamido)-7-(2-triphenylphosphonioethoxy)cephalosporanatebromide

Benzhydryl 7-(2-thienylacetamido)-7-(2-bromoethoxy)cephalosporanate (673mg.) is refluxed in 15 ml. benzene with 262 mg. triphenylphosphineovernight. Crude benzhydryl7-(2-thienylacetamido)-7-(2-triphenylphosphonoethoxy)cephalosporanatebromide is obtained on evaporation of the benzene and is used directlyfor the next step.

D. Benzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate

The sample of benzhydryl7-(2-thienylacetamido)-7-(2-triphenylphosphonoethoxy)cephalosporanatebromide obtained from the previous step (ca. 1 mmole) is stirred undernitrogen at -30° C. in 50 ml. of ether. An equivalent amount ofphenyllithium in ether is slowly added over 30 minutes. The reactionmixture is then allowed to warm with stirring, over about 1/2 hour, to0° C. The reaction mixture is quenched with 50 ml. of 0.1 M aqueous pH 7phosphate buffer at 0° C. The ether layer is separated and the aqueousphase washed twice more with ether. The combined organic portions aredried over magnesium sulfate, filtered, evaporated and chromatographedon silica gel, eluting with B 3% methanol in chloroform, to affordbenzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate afterevaporation of the solvent under reduced pressure.

E. Sodium 7-(2-thienylacetamido)-7-hydroxycephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate (566 mg.) isdissolved in 0.8 g. anisole and cooled to 0° C. Trifluoroacetic acid (4ml.) precooled to 0° C. is added and the reaction allowed to proceed for2 minutes at 0° C. Vacuum of 0.1 mm. is immediately applied and thereaction mixture allowed to warm to room temperature without externalheating. Anisole is then distilled out at 30° C./0.1 mm. A few ml.anisole is added to the residue and evaporated at 30° C./0.1 mm. toinsure total removal of trifluoroacetic acid. The product is treatedwith 5 ml. of water containing 84 mg. of sodium bicarbonate andlyophilized. The resulting powder is washed thoroughly with ether anddried, affording sodium7-(2-thienylacetamido)-7-hydroxycephalosporanate.

EXAMPLES 35-39

When Example 34, step B above is carried out using phenylaceticanhydride, acetic anhydride, benzothiophene-2-acetic anhydride,furylacetic anhydride or tetraazolylacetic anhydride in place ofthienylacetic anhydride, the corresponding benzhydryl7-acylamido-7-(2-bromoethoxy)cephalosporanate is obtained. Theseintermediate products, when treated by the procedures described in C, Dand E above, afford respectively:

    ______________________________________                                        Example No.                                                                            Product                                                              ______________________________________                                        35       Sodium 7-benzylamido-7-hydroxycephalosporanate                       36       Sodium 7-acetamido-7-hydroxycephalosporanate                         37       Sodium 7-(2-thianaphthene-2-acetamido)-7-                                     hydroxycephalosporanate                                              38       Sodium 7-(2-furylacetamido)-7-hydroxy-                                        cephalosporanate                                                     39       Sodium 7-tetraazolylacetamido-7-hydroxy-                                      cephalosporanate                                                     ______________________________________                                    

EXAMPLE 40 A. Benzhydryl7-(2-thienylacetamido)-7-(aminocarbonyloxy)cephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate (566 mg.) isdissolved in 30 ml. of ether and treated at 0° C. with 80 mg.aminocarbonyl chloride and 0.3 ml. pyridine. After 15 minutes, thesolution is washed successively with water, dilute aqueous phosphoricacid (buffered to pH 2), water, and aqueous sodium bicarbonate. Afterdrying over magnesium sulfate, filtration and evaporation of thesolvent, the residue is essentially pure benzhydryl7-(2-thienylacetamido)-7-(aminocarbonyloxyl)cephalosporanate.

B. Sodium 7-(2-thienylacetamido)-7-(aminocarbonyloxy)cephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-(aminocarbonyloxy)cephalosporanateis deblocked following the procedures described in Example 34E above toafford sodium7-(2-thienylacetamido)-7-(aminocarbonyloxy)cephalosporanate.

EXAMPLE 41 A. Benzhydryl7-(2-thienylacetamido)-7-(methoxycarbonyloxy)cephalosporanate

To benzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate in 30 ml.of ether is added 95 mg. of methyl chlorocarbonate and 0.3 ml. ofpyridine. After 15 minutes the reaction mixture is worked up asdescribed in Example 40A to afford benzhydryl7-(2-thienylacetamido)-7-(methoxycarbonyloxy)cephalosporanate.

B. Sodium 7-(2-thienylacetamido)-7-(methoxycarbonyloxy)cephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-(methoxycarbonyloxy)cephalosporanateis deblocked following the procedures described in Example 34E above toafford sodium7-(2-thienylacetamido)-7-(methoxycarbonyloxy)cephalosporanate.

EXAMPLE 42 A. Benzhydryl7-(2-thienylacetamido)-7-(aminosulfonyloxy)cephalosporanate

To benzhydryl 7-(2-thienylacetamido)-7-hydroxycephalosporanate in 30 ml.of ether is added 116 mg. of aminosulfonyl chloride and 0.3 ml. ofpyridine. After 15 minutes the reaction mixture is worked up asdescribed in Example 40A to afford benzhydryl7-(2-thienylacetamido)-7-(aminosulfonyloxy)cephalosporanate.

B. Sodium 7-(2-thienylacetamido)-7-(aminosulfonyloxy)cephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-(aminosulfonyloxy)cephalosporanateis deblocked following the procedures described in Example 34E above toafford sodium7-(2-thienylacetamido)-7-(aminosulfonyloxy)cephalosporanate.

EXAMPLE 43 A. 7-(p-Nitrobenzylideneamino)cephalosporanic acid benzhydrylester

7-Aminocephalosporanic acid benzhydryl ester (438 mg.) is refluxed 1hour in 50 ml. benzene with 151 mg. p-nitrobenzaldehyde in an azeotropicdrying apparatus. The solvent is vacuum distilled away, leaving 571 mg.crystalline product. If desired, it can be recrystallized frombenzenecylohexane (1:2) to obtain the product in pure form.

B. Benzhydryl 7-(p-nitrobenzylideneamino)-7-acetoxycephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate (571 mg.) isstirred at 0° C. under nitrogen in 10 ml. acetonitrile. Acetyl peroxide(118 mg.) is introduced, followed by 129 mg. diisopropylethylamine,which is added in acetonitrile over a 5 minute period.

The reaction mixture is aged 5 minutes at room temperature andevaporated under reduced pressure. The residue is taken up in 25 ml. ofbenzene and washed successively with water, dilute phosphoric acid(buffered at pH 2) water and aqueous sodium bicarbonate. The solution isdried over magnesium sulfate, filtered, evaporated and chromatographedon silica gel, using 4:1 chloroform-ethylacetate to elute the product.Evaporation of the solvent affords benzhydryl7-(p-nitrobenzylideneamino)-7-acetoxycephalosporanate.

C. Benzhydryl 7-amino-7-acetoxycephalosporanate hydrochloride

Benzhydryl 7-(p-nitrobenzylideneamino)-7-acetoxycephalosporanate (629mg.) and 260 mg. aniline hydrochloride are stirred together for 1 hourat 25° C. in 10 ml. methanol. The methanol is removed at 0.1 mm.pressure and 30° C. and the residue covered with ether to crystallizefor 1 hour. The solid is triturated with ether, filtered, and washedwith ether several times. It consists of benzhydryl7-amino-7-acetoxycephalosporanate hydrochloride and anilinehydrochloride mixed together and is used immediately in the next step.

D. Benzhydryl 7-(2-thienylacetamido)-7-acetoxycephalosporanate

The mixture of benzhydryl 7-amino-7-acetoxycephalosporanatehydrochloride and aniline hydrochloride obtained in the previousexperiment is stirred vigorously at -10° C. in 25 ml. methylenechloride. Thienylacetyl chloride (0.5 g.) is added, and then 0.5 g.triethylamine is slowly introduced. This liberates the free 7-aminocompound which is instantly acylated. The reaction mixture is slowlyallowed to warm to room temperature. Excess acid chloride is hydrolyzedby shaking with water, and the methylene chloride layer is then washedsuccessively with dilute phosphoric acid (buffered to pH 2), water anddilute aqueous sodium bicarbonate. After drying over magnesium sulfate,the solution is filtered and evaporated, affording a mixture of theproduct and N-phenyl thienylacetamide. Chromatography over silica geland elution with 4:1 chloroform-ethylacetate affords the title compound.

E. Sodium 7-(2-thienylacetamido)-7-acetoxycephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-acetoxycephalosporanate (620 mg.) isdissolved in 0.8 ml. anisole and cooled to 0° C. Trifluoroacetic acid (4ml.) precooled to 0° C. is added and the reaction allowed to proceed for2 minutes at 0° C. Vacuum of 0.1 mm. is immediately applied and thereaction mixture allowed to warm to room temperature. Anisole is thendistilled at 30° C./0.1 mm. A few ml. anisole is added to the residueand pulled off at 30° C./0.1 mm. to insure total removal oftrifluoroacetic acid. The product is treated with 5 ml. water containing84 mg. of sodium bicarbonate and lyophilized. The resulting powder iswashed thoroughly with ether and dried, affording the title compound.

EXAMPLE 44 A. p-Methoxybenzyl 7-azido-7-bromocephalosporanate

A mixture of p-methoxybenzyl 7-aminocephalosporanate (3.9 g.) in 250 ml.methylene chloride and sodium nitrite (1.4 g.) in 250 ml. of water isstirred under nitrogen with ice cooling and 7.5 ml. of 2 N sulfuric acidis added all at once. The mixture is stirred at 3° C. for 1 hour and theorganic phase is separated and rapidly dried over sodium sulfate andconcentrated to 50 ml. in vacuo. To this solution ofp-methoxybenzyl-7-diazocephalosporanate cooled to -40° C. is rapidlyadded 50 ml. of triethylammonium azide solution and 50 ml. of bromineazide solution prepared as described below. The solution is allowed towarm to 0° C., then washed with 200 ml. of 2% sodium hydrogen phosphatesolution. The orgaic phase is dried over sodium sulfate and evaporated.The residue is rapidly chromatographed on a 4.5 cm.×15 cm. column ofsilica gel using 20% methylene chloride 80% benzene as eluant, affordingp-methoxybenzyl 7-azido-7-bromocephalosporanate.

Preparation of Triethylammonium Azide Solution

To a slurry of 6 g. of sodium azide in 10 ml. of water and 100 ml. ofmethylene chloride at -10° C. is added 10 ml. of 50% H₂ SO₄. The organicphase is decanted from the aqueous paste and dried over anhydrousmagnesium sulfate. To 50 ml. of this hydrazoic acid solution is added2.25 ml. of triethylamine. The resulting pH is 7.

Preparation of Bromine Azide Solution

To 50 ml. of the above hydrazoic acid solution is added 2 g. ofN-bromosuccinimide with stirring and ice cooling. After 10 minutes at 0°C. the reaction is complete and the solution is used immediately forreaction with 7-diazocephalosporanates.

The p-methoxybenzyl 7-aminocephalosporanate used as the startingmaterial is prepared from 7-aminocephalosporanic acid using theprocedures described in Example 19A, B and C for the preparation ofp-methoxybenzyl 3-methyl-7-aminodecephalosporanate.

B. p-Methoxybenzyl 7-azido-7-(2-methoxyethoxy)cephalosporanate

To a solution of 4.8 g. of p-methoxybenzyl7-azido-7-bromocephalosporanate in 20 ml. of 2-methoxyethanol is added1.95 g. of silver tetrafluoroborate and 0.8 ml. of pyridine. Afterstirring for 3 hours at room temperature, the 2-methoxyethanol isremoved under vacuum. The residue is taken up in methylene chloride,filtered, and the filtrate washed with water, dried and evaporated. Theresidue is chromatographed on 140 g. of silica gel. Elution with 2%methanol in chloroform gives substantially pure p-methoxybenzyl7-azido-7-(2-methoxyethoxy)cephalosporanate.

C. p-Methoxybenzyl 7-amino-7-(2-methoxyethoxy)cephalosporanate

p-Methoxybenzyl 7-azido-7-(2-methoxyethoxy)cephalosporanate (2 g.) andplatinum oxide (2 g.) in 200 ml. of dioxane are vigorously stirred underan atmosphere of hydrogen for 1 hour. Fresh catalyst (2 g.) is added andthe hydrogenation continued for 2 hours. The dioxane is evaporated underreduced pressure and the catalyst adsorbed onto 30 g. of silica gel in50 ml. of chloroform and filtered. The cake is washed with 500 ml. ofchloroform and the combined filtrate and washings evaporated givingp-methoxybenzyl 7-amino-7-(2-methoxyethoxy)cephalosporanate as a viscousoil.

D. p-Methoxybenzyl7-(D-α-azidophenylacetamido)-7-(2-methoxyethoxy)cephalosporanate

To an ice-cooled solution of 0.9 g. of p-methoxybenzyl7-amino-7-(2-methoxyethoxy)cephalosporanate in 20 ml. of methylenechloride is added 0.8 g. of D-α-azidophenylacetyl chloride and 0.7 ml.of dry pyridine. The solution is stirred at 2° C. for 20 minutes, thenpoured into 20 ml. of water. The mixture is acidified to pH 2 withdilute sulfuric acid and the organic phase is separated, washed withwater, dried and evaporated. The residue is chromatographed on 50 g.silica gel. Elution with chloroform-ethylacetate yields p-methoxybenzyl7-(D-α-azidophenylacetamido)-7-(2-methoxyethoxy)cephalosporanate.

E. 7-(D-α-Azodiphenylacetamido)-7-(2-methoxyethoxy)cephalosporanic acid

A solution of 420 mg. of p-methoxybenzyl7-(D-α-azidophenylacetamido)-7-(2-methoxyethoxy)cephalosporanate, 300mg. of anisole and 3 ml. of trifluoroacetic acid in 10 ml. of benzene isstirred for 2 hours at room temperature. The reaction mixture isevaporated and the residue is taken up in ethylacetate and extractedwith sodium bicarbonate solution. The aqueous extract is cooled to 0°C., overlayered with ethylacetate and acidified to pH 2.1 with dilutesulfuric acid. The ethylacetate layer is separated, dried over anhydroussodium sulfate and evaporated, affording7-(D-α-azidophenylacetamido)-7-(2-methoxyethoxy)cephalosporanic acid.

F. 7-(D-α-aminophenylacetamido)-7-(2-methoxyethoxy)cephalosporanic acid

To an ice-cooled solution of 240 mg. of7-(D-α-azidophenylacetamido)-7-(2-methoxyethoxy)cephalosporanic acid in3 ml. of acetic acid and 2 ml. of water is added 1.5 g. of powderedzinc. The mixture is stirred for 15 minutes, then filtered. The residueis washed with 10 ml. of water and the combined filtrate and washingsare evaporated to dryness. The residue is taken up in 2 ml. of water andpoured onto a 2×40 cm. column of ion retardation resin. The column iseluted with water and the ninhydrin positive fractions lyophilized,giving 7-(D-α-aminophenylacetamido)-7-(2-methoxyethoxy)cephalosporanicacid as a yellow powder.

EXAMPLE 45 A. p-Methoxybenzyl7-(2-methoxyethoxy)-7-(2-thienylacetamido)cephalosporanate

Following the procedure described in Example 3B, an equivalent quantityof p-methoxybenzyl 7-amino-7-(2-methoxyethoxy)cephalosporanate isreacted with 2-thienylchloride and the resulting reaction product isworked up in the same manner to afford p-methoxybenzyl7-(2-methoxyethoxy)-7-(2-thienylacetamido)cephalosporanate.

B. Sodium 7-(2-methoxyethoxy)-7-(2-thienylacetamido)cephalosporanate

A solution of 0.4 g. of p-methoxybenzyl7-(2-methoxyethoxy)-7-(2-thienylacetamido)cephalosporanate is dissolvedin 3.5 ml. of anisole and treated with 10 ml. of trifluoroacetic acid atroom temperature for 10 minutes. The trifluoroacetic acid and anisoleare removed under reduced pressure maintaining the temperature below 40°C., and the residue is taken up in 25 ml. of chloroform and treated with20 ml. of water containing 0.120 g. of sodium bicarbonate. The mixtureis stirred for 1/2 hour at room temperature and the organic phase isseparated and washed with water. The combined aqueous phase is washedtwice with methylene chloride and lyophilized affording sodium7-(2-methoxyethoxy)-7-(2-thienylacetamido)cephalosporanate.

EXAMPLES 46-50

In similar manner as described in Example 45, p-methoxybenzyl7-amino-7-(2-methoxyethoxy)cephalosporanate is reacted with2-thianaphthene-2-acetyl chloride, p-guanidinophenylacetyl chloridehydrochloride, furylacetyl chloride, phenylacetyl chloride, ortetraazolylacetyl chloride, and the resulting ester is cleaved to obtain

    ______________________________________                                        Example   Product                                                             ______________________________________                                        46        Sodium 7-(2-methoxyethoxy)-7-(2-thianaphthene-                                2-acetamido)cephalosporanate                                        47        7-(2-methoxyethoxy)-7-(p-guanidinophenyl-                                     acetamido)cephalosporanic acid                                      48        Sodium 7-(2-methoxyethoxy)-7-(2-furylacet-                                    amido)cephalosporanate                                              49        Sodium 7-(2-methoxyethoxy)-7-phenylacetamido-                                 cephalosporanate                                                    50        Sodium 7-(2-methoxyethoxy)-7-tetraazolyl-                                     acetamidocephalosporanate                                           ______________________________________                                    

EXAMPLE 51 A. Benzhydryl7-azido-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate

A mixture of 3.1 g. of benzhydryl 7-azido-7-bromocephalosporanate, 1.1.g. of silver fluoroborate, 2 g. of t-butoxycarbonylglycine and 0.45 ml.of pyridine in 10 ml. of dioxane is stirred under the exclusion ofmoisture and light for 3 hours at room temperature. The mixture isdiluted with 100 ml. of ether and filtered and the filtrate is washedsuccessively with 2% aqueous phosphoric acid, water and 5% aqueoussodium bicarbonate solution. The ethereal phase is dried over anhydroussodium sulfate and evaporated and the residue is chromatographed on 140g. of silica gel. Elution with 2% methanol in chloroform givesbenzhydryl 7-azido-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate.

B. Benzhydryl 7-amino-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate

A solution of 3 g. of benzhydryl7-azido-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate in 300 ml. ofdry dioxane is hydrogenated in the presence of 3 g. Pt oxide for 1 hourat room temperature. Fresh catalyst (3 g.) is added and thehydrogenation continued for 3 hours. The dioxane is evaporated underreduced pressure and the residue taken up in chloroform and filteredthrough 20 g. of silica gel packed in a fritted glass funnel. The cakeis washed with 500 ml. of chloroform and the combined washings andfiltrate are evaporated, leaving benzhydryl7-amino-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate.

C. Benzhydryl7-(t-butoxycarbonylaminoacetoxy)-7-(2-furylacetamido)cephalosporanate

To a cooled solution of 2 g. of benzhydryl7-amino-7-(t-butoxycarbonylaminoacetoxy)cephalosporanate in 30 ml. ofmethylene chloride is added 1.5 ml. of 2-furylacetyl chloride and 1.5ml. of dry pyridine. The mixture is stirred at 0° C. for 15 minutes,then poured over ice. The methylene chloride layer is separated, washedsuccessively with 2% phosphoric acid, water and 2% sodium bicarbonatesolution, dried and evaporated. The residue is chromatographed on 80 g.of silica gel. Elution with chloroformethylacetate mixtures yieldssubstantially pure benzhydryl7-(t-butoxycarbonylaminoacetoxy)-7-(2-furylacetamido)cephalosporanate.

D. 7-aminoacetoxy-7-(2-furylacetamido)cephalosporanic acidtrifluoroacetate salt

A solution of 400 mg. of benzhydryl7-(t-butoxycarbonylaminoacetoxy)-7-(2-furylacetamido)cephalosporanate in3 ml. of anisole and 3 ml. of trifluoroacetic acid is kept at roomtemperature for 5 minutes, then the excess trifluoroacetic acid ispumped off under high vacuum for 5 minutes. The residue is trituratedwith ether and the resulting powder filtered off and washed with ether.The residue is dissolved in 200 ml. of water and filtered through 50 mg.of charcoal layered on diatomaceous earth. The filtrate is lyophilizedaffording 7-aminoacetoxy-7-(2-furylacetamido)cephalosporanic acidtrifluoroacetate salt as a light powder.

The zwitterionic inner salt is obtained by neutralizing an aqueoussolution to pH 6.5 followed by concentration to a small volume andprecipitation with alcohol.

EXAMPLE 52

When the process of Example 51 is repeated using tetraazolylacetylchloride, 2-thienylacetyl chloride or phenylacetyl chloride in place of2-furylacetyl chloride, the corresponding 7-tetraazolylacetamido,7-(2-thienylacetamido), or7-phenylacetamido-7-aminoacetoxycephalosporanic acid trifluoroacetatesalt is obtained.

EXAMPLE 53 A. Benzhydryl 7-azido-7-benzyloxycephalosporanate

A mixture of 4.6 g. of benzhydryl 7-azido-7-bromocephalosporanate, 1.6g. of silver fluoroborate and 0.66 ml. of dry pyridine in 15 ml. ofbenzyl alcohol is stirred at room temperature for 21/2 hours. Themixture is diluted with 300 ml. of ether and filtered. The filtrate iswashed with 2% phosphoric acid and with water, then dried over sodiumsulfate and evaporated. The excess benzyl alcohol is removed on a highvacuum pump with magnetic stirring for 18 hours and the residuechromatographed on 200 g. of silica gel. Elution with hexane followed byincreasing concentrations of methylene chloride in hexane affordsbenzhydryl 7-azido-7-benzyloxycephalosporanate.

B. Benzhydryl 7-amino-7-benzyloxycephalosporanate

A solution of 3 g. of benzhydryl 7-azido-7-benzyloxycephalosporanate in300 ml. of dry dioxane is hydrogenated in the presence of 3 g. ofplatinum oxide at room temperature at atmospheric pressure for 1 hour.Fresh catalyst (3 g.) is added and the hydrogenation continued for 2hours. The dioxane is removed under reduced pressure and the residuetaken up in chloroform and filtered through 20 g. of silica gel packedin a fritted glass funnel. The cake is washed with 500 ml. of chloroformand the combined filtrate and washings are evaporated, leavingbenzhydryl 7-amino-7-benzyloxycephalosporanate as a yellow resin.

C. Benzhydryl 7-benzyloxy-7-(2-thienylacetamido)cephalosporanate

To a cooled solution of 1.2 g. of benzhydryl7-amino-7-benzyloxycephalosporanate in 15 ml. of methylene chloride isadded with stirring 0.8 ml. of 2-thienylacetyl chloride and 0.8 ml. ofpyridine. The mixture is stirred for 15 minutes at 2° C. and then pouredover ice. The organic phase is separated and washed successively with 2%phosphoric acid, water and 2% sodium bicarbonate solution, then driedover anhydrous sodium sulfate and evaporated. The residue ischromatographed on 50 g. of silica gel with 5% ethylacetate in methylenechloride as eluant, giving benzhydryl7-benzyloxy-7-(2-thienylacetamido)cephalosporanate.

D. Sodium 7-benzyloxy-7-(2-thienylacetamido)cephalosporanate

A solution of 0.9 g. of benzhydryl7-benzyloxy-7-(2-thienylacetamido)cephalosporanate in 3 ml. of anisoleand 7 ml. of trifluoroacetic acid is stirred at room temperature for 5minutes and the excess anisole and trifluoroacetic acid rapidly removedon the vacuum pump. The residue is taken up in a mixture of 25 ml. ofmethylene chloride and 25 ml. of water and rapidly stirred while sodiumbicarbonate is added to bring the pH to 6.5. The aqueous layer isseparated, washed once with ether and lyophilized, giving sodium7-benzyloxy-7-(2-thienylacetamido)cephalosporanate as a light powder.

EXAMPLE 54 Sodium 7-hydroxy-7-(2-thienylacetamido)cephalosporanate

A solution of sodium 7-benzyloxy-7-(2-thienylacetamido)cephalosporanate(300 mg.) in 10 ml. of water is hydrogenated at 40 PSIG and roomtemperature in the presence of 0.39 g. of 10% palladium-on-charcoal for6 hours. Fresh catalyst (0.3 g.) is added and the hydrogenationcontinued for 18 hours. The catalyst is filtered and the filtratelyophilized, leaving sodium7-hydroxy-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 55

Following the procedures described in Example 53 using furylacetylchloride, p-guanidinophenylacetyl chloride hydrochloride,tetraazolylacetyl chloride, or phenylacetyl chloride in place of2-thienylacetyl chloride, there are obtained, respectively,

sodium 7-benzyloxy-7-(2-furylacetamido)cephalosporanate,

7-benzyloxy-7-(p-guanidinophenylacetamido)cephalosporanic acid,

sodium 7-benzyloxy-7-tetraazolylacetamidocephalosporanate, and

sodium 7-benzyloxy-7-phenylacetamido-cephalosporanate.

EXAMPLE 56

Following the procedures described in Example 54, the7-benzyloxycephalosporins are converted to the corresponding7-hydroxycephalosporins.

EXAMPLE 57 A. Benzhydryl7-azido-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanate

The benzhydryl ester of N-t-butoxycarbonyl-L-serine is prepared by theaddition of a solution of diphenyldiazomethane (2.5 g.) in 10 ml. ofdioxane to a solution of N-t-butoxycarbonyl-L-serine (2.5 g.) in 5 ml.of dioxane. To the resulting solution is added benzhydryl7-azido-7-bromo-cephalosporanate (4.5 g.), silver fluoroborate (1.6 g.)and pyridine (0.65 ml.). The mixture is stirred for 3 hours at roomtemperature, then diluted with 100 ml. of ether and filtered. Thefiltrate is washed successively with pH 2 phosphoric acid buffer, waterand sodium bicarbonate solution and then dried over anhydrous sodiumsulfate and evaporated. The residue is chromatographed on 200 g. ofsilica gel. Elution with chloroform-ethylacetate mixture affords theproduct as a tan gum.

B. Benzhydryl7-amino-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanate

A solution of 3 g. of benzhydryl7-azido-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanate in 300 ml. of dry dioxane is hydrogenated in thepresence of 3 g. of platinum oxide at room temperature and atmosphericpressure for 1 hour. Fresh catalyst (3 g.) is added and thehydrogenation continued for 2 hours. The dioxane is removed underreduced pressure and the residue taken in chloroform and filteredthrough 20 g. of silica gel packed in a fritted glass funnel. The cakeis washed with chloroform and the combined washings and filtrate areevaporated, leaving benzhydryl7-amino-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanate.

C. Benzhydryl7-(α-benzhydryloxycarbonylphenylacetamido)-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)-cephalosporanate

α-Benzhydryloxycarbonylphenylacetyl chloride (377 mg.) is added in oneportion to a cooled solution of 450 mg. of benzhydryl7-amino-7-(L-2-benzhydryloxycarbonyl2-t-butoxycarbonylaminoethoxy)cephalosporanateand 0.2 ml. of dry pyridine in 10 ml. of methylene chloride. Thesolution is stirred for 20 minutes at 0° C., then poured into 20 ml. ofpH 2 phosphoric acid buffer. The organic phase is washed with water andsodium bicarbonate solution, dried over anhydrous sodium sulfate andevaporated. The residue is chromatographed on 30 g. of silica gel.Elution with chloroform yields benzhydryl7-(α-benzhydryloxycarbonylphenylacetamido)-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanate.

D. Disodium7-(L-2-carboxy-2-aminoethoxy)-7-(α-carboxyphenylacetamido)cephalosporanate

2 ml. of anisole and 6 ml. of trifluoroacetic acid are added to 200 mg.of benzhydryl7-(α-benzhydryloxycarbonylphenylacetamido)-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanateand the mixture is stirred at room temperature for 8 minutes. Excessanisole and trifluoroacetic acid are rapidly evaporated on a vacuumpump. The residue is taken up in 20 ml. of methylene chloride and 20 ml.of water and the mixture vigorously stirred as the pH is adjusted to 7by the addition of sodium bicarbonate solution. The aqueous phase isseparated, washed once with ether and lyophilized, giving the disodiumsalt of7-(L-2-carboxy-2-aminoethoxy)-7-(α-carboxyphenylacetamido)cephalosporanate.

In a similar manner starting with N-t-butoxycarbonyl-D-serine in placeof N-t-butoxycarbonyl-L-serine,7-(D-2-carboxy-2aminoethoxy)-7-(α-carboxyphenylacetamido)cephalosporanateis obtained.

EXAMPLE 58

When benzhydryl7-amino-7-(L-2-benzhydryloxycarbonyl-2-t-butoxycarbonylaminoethoxy)cephalosporanateis acylated with phenylacetyl chloride, 2-thienylacetyl chloride orfurylacetyl chloride in accordance with the procedures described in theforegoing examples, the corresponding 7-phenylacetamido,7-(2-thienylacetamido) and 7-(2-furylacetamido) analogs are obtained.

EXAMPLE 59 A. Benzhydryl 7-azido-7-(carbamoylmethoxy)cephalosporanate

To a solution of 3.1 g. of benzhydryl 7-azido-7-bromocephalosporanateand 2 g. of glycolamide in 10 ml. of dioxane is added 1.1 g. of silverfluoroborate and 0.45 ml. of pyridine. The mixture is stirred for 2hours at room temperature, then diluted with 100 ml. of ether andfiltered. The filtrate is washed successively with 2% aqueous phosphoricacid, water and sodium bicarbonate solution, then dried over anhydroussodium sulfate and evaporated. The residue is chromatographed on 120 g.of silica gel. Elution with 2% methanol in chloroform affordssubstantially pure benzhydryl7-azido-7-(carbamoylmethoxy)cephalosporanate.

B. Benzhydryl 7-amino-7-(carbamoylmethoxy)cephalosporanate

A solution of 3 g. of benzhydryl7-azido-7-(carbamoylmethoxy)cephalosporanate in 300 ml. of dry dioxaneis vigorously stirred with 3 g. of platinum oxide under one atmosphereof hydrogen at room temperature for 1 hour. Fresh catalyst (3 g.) isadded and the hydrogenation continued for 2 hours. The dioxane isremoved under reduced pressure and the residue is taken up in chloroformand filtered through 20 g. of silica gel packed in a sintered glassfunnel. The silica gel is washed with chloroform and the combinedfiltrate and washes are evaporated, leaving benzhydryl7-amino-7-(carbamoylmethoxy)cephalosporanate.

C. Benzhydryl 7-carbamoylmethoxy-7-(2-thienylacetamido)cephalosporanate

To a cooled solution of 1.5 g. of benzhydryl7-amino-7-(carbamoylmethoxy)cephalosporanate in 20 ml. of methylenechloride is added 1.0 g. of thienylacetyl chloride and 1.0 ml. of drypyridine. The mixture is stirred for 15 minutes at 0°-2° C. and thenpoured into 100 ml. of pH 2 phosphoric acid buffer. The methylenechloride phase is washed with water and sodium bicarbonate solution,dried over anhydrous sodium sulfate and evaporated. The product ischromatographed over 50 g. of silica gel with 5% ethylacetate-chloroformas eluant, affording benzhydryl7-carbamoylmethoxy-7-(2-thienylacetamido) cephalosporanate.

D. Sodium 7-carbamoylmethoxy-7-(2-thienylacetamido)cephalosporanate

A mixture of 0.5 g. of benzhydryl7-carbamoylmethoxy-7-(2-thienylacetamido)cephalosporanate in 3 ml. ofanisole and 7 ml. of trifluoroacetic acid is stirred at 0° C. for 10minutes. Excess anisole and trifluoroacetic acid are rapidly evaporatedon a vacuum pump. The residue is taken up in 40 ml. of water and 40 ml.of methylene chloride and the mixture vigorously stirred as the pH isadjusted to 6.5 by the addition of sodium bicarbonate. The aqueous phaseis separated, washed once with ether and lyophilized, giving sodium7-carbamoylmethoxy-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 60

When benzhydryl 7-amino-7-(carbamoylmethoxy)cephalosporanate is acylatedwith phenylacetyl chloride, furylacetyl chloride or tetraazolylacetylchloride in place of 2-thienylacetyl chloride, the corresponding sodium7-phenylacetamido, 7-(2-furylacetamido) and7-tetraazolylacetamido-7-(carbamoylmethoxy)cephalosporins are obtained.

EXAMPLE 61 A. Benzhydryl7-azido-7-(benzhydryloxycarbonylmethoxy)cephalosporanate

A solution of the benzhydryl ester of glycolic acid is prepared by theaddition of a solution of 2.5 g. of diphenyldiazomethane in 10 ml. ofdioxane to a solution of 0.7 g. of glycolic acid in 5 ml. of dioxane. Tothis is added 4.5 g. of benzhydryl 7-azido-7-bromocephalosporanate, 1.6g. of dry silver fluoroborate and 0.65 ml. of dry pyridine. The mixtureis stirred for 3 hours at room temperature, then diluted with ether andfiltered. The filtrate is washed with water, dried over anhydrous sodiumsulfate an evaporated. The residue is chromatographed over 160 g. ofsilica gel, affording benzhydryl7-azido-7-(benzhydryloxycarbonylmethoxy)cephalosporanate.

B. Benzhydryl 7-amino-7-(benzhydryloxycarbonylmethoxy)cephalosporanate

A solution of 3 g. of benzhydryl7-azido-7-(benzhydryloxycarbonylmethoxy)cephalosporanate in 300 ml. ofdry dioxane is vigorously stirred with 3 g. of platinum oxide under oneatmosphere of hydrogen at room temperature for 1 hour. Fresh catalyst (3g.) is added and the hydrogenation continued for 2 hours. The dioxane isremoved under reduced pressure and the residue taken up in chloroformand filtered through 20 g. of silica gel packed in a sintered glassfunnel. The silica gel is washed with 300 ml. of chloroform and thefiltrate and washings combined and evaporated, leaving benzhydryl7-amino-7-(benzhydryloxycarbonylmethoxy)cephalosporanate as a heavy oil.

C. Benzhydryl 7-(benzhydryloxycarbonylmethoxy)cephalosporanate

To a cooled solution of 0.4 g. of benzhydryl7-amino-7-(benzhydryloxycarbonylmethoxy)cephalosporanate in 5 ml. ofmethylene chloride is added 0.25 g. of thienylacetyl chloride and 0.25ml. of pyridine. The mixture is stirred for 15 minutes at 0°-3° C. andthen poured over ice. The organic phase is washed successively with 2%phosphoric acid, water and aqueous sodium bicarbonate solution andfinally dried over anhydrous sodium sulfate and evaporated. The residueis chromatographed over 25 g. of silica gel of the product eluted with5% ethylacetate in methylene chloride, affording benzhydryl7(benzhydryloxycarbonylmethoxy)-7-(2-thienylacetamido)cephalosporanate.

D. Disodium 7-(carboxymethoxy)-7-(2-thienylacetamido)cephalosporanate

To a solution of 0.4 g. of benzhydryl7-(benzhydryloxycarbonylmethoxy)-7-(2-thienylacetamido)cephalosporanatein 3 ml. of anisole is added 5 ml. of trifluoroacetic acid. The mixtureis kept for 8 minutes, then the excess anisole and trifluoroacetic acidare rapidly evaporated on a vacuum pump. The residue is stirred in amixture of 20 ml. of methylene chloride and 20 ml. of water and the pHadjusted to 6.5 (glass electrode) with sodium bicarbonate. The aqueousphase is separated and extracted once with ether and then lyophilized,affording the disodium salt of7-(carboxymethoxy)-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 62

When benzhydryl 7-amino-7-(benzhydryloxycarbonylmethoxy)cephalosporanateis reacted with 2-thianaphthene-2-acetyl chloride or monobenzhydrylphenylmalonyl chloride in place of thienyl chloride and the resultingreaction product is recovered and then deblocked following theprocedures of Example 61, sodium7-(2-carboxymethoxy)-7-(2-thianaphthene-2-acetamido)cephalosporanate andsodium7-(2-carboxymethoxy)-7-(2-carboxy-2-phenylacetamido)cephalosporanate areobtained.

EXAMPLE 63 A. Benzhydryl 7-acetoxy-7-azidocephalosporanate

A mixture of 2.2 g. of benzhydryl 7-azido-7-bromocephalosporanate and0.8 g. of silver acetate in 10 ml. of acetic acid is stirred at roomtemperature for 3 hours. The acetic acid is removed under reducedpressure and the residue taken up in methylene chloride and filtered.The filtrate is washed with sodium bicarbonate solution, then dried oversodium sulfate and evaporated. The residue is chromatographed over 80 g.of silica gel. Elution with chloroform yields benzhydryl7-acetoxy-7-azidocephalosporanate.

B. Benzhydryl 7-amino-7-acetoxycephalosporanate

2 g. of benzhydryl 7-acetoxy-7-azidocephalosporanate in 200 ml. of drydioxane is hydrogenated at room temperature and atmospheric pressure inthe presence of 2 g. of platinum oxide for 1 hour. Fresh catalyst (2 g.)is added and the hydrogenation continued for 2 hours. The solvent isevaporated and the residue is dissolved in ether and shaken with 10 ml.of powdered anhydrous magnesium sulfate and filtered throughdiatomaceous earth in a fritted glass funnel. The filtrate isevaporated, leaving benzhydryl 7-amino-7-acetoxycephalosporanate.

C. Benzhydryl 7-acetoxy-7-(2-thienylacetamido)cephalosporanate

To a cooled solution of 1.2 g. of benzhydryl7-amino-7-acetoxycephalosporanate in 20 ml. of methylene chloride isadded 0.8 ml. of thienylacetyl chloride and 0.8 ml. of pyridine. Themixture is stirred at 0° C. for 15 minutes then poured over ice. Theorganic phase is washed successively with 2% phosphoric acid solution,water and 2% sodium bicarbonate solution, dried over anhydrous sodiumsulfate and evaporated. The residue is chromatographed on 50 g. ofsilica gel. Elution with chloroform yields benzhydryl7-acetoxy-7-(2-thienylacetamido)cephalosporanate.

D. Sodium 7-acetoxy-7-(2-theinylacetamido)cephalosporanate

A solution of 2 g. of benzhydryl7acetoxy-7-(2-thienylacetamido)cephalosporanate in 8 ml. of anisole and16 ml. of trifluroroacetic acid is stirred at room temperature for 5minutes. The excess anisole and trifluoroacetic acid are rapidly removedon the vacuum pump and the residue is taken up in 50 ml. of methylenechloride and extracted twice with sodium bicarbonate solution. Theaqueous extract is cooled and overlayered with ethylacetate and the pHadjusted to 2.1 with dilute sulfuric acid. The ethylacetate layer isvigorously stirred with water as the pH is adjusted to 6.5 (glasselectrode). Evaporation of the aqueous extract gives the sodium salt of7-acetoxy-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 64 Sodium3-hydroxymethyl-7-hydroxy-7-(2-thienylacetamido)decephalosporanate

1.8 g. of sodium 7-acetoxy-7-(2-thienylacetamido)cephalosporanate isdissolved in 50 ml. of citrus acetylesterase solution heated in a waterbath at 30° C. The pH is maintained at 6.6 by the addition of N sodiumhydroxide solution under the control of a pH stat. After 8 hours 15 g.of sodium chloride is added, the solution is overlayered with 100 ml. ofethylacetate and the pH taken to 2.1 with hydrochloric acid. The mixtureis centrifuged and the supernatant ethylacetate separated. Theethylacetate extract is vigorously stirred with water as the pH is takento 7 with sodium bicarbonate solution. The aqueous phase is separatedand lyophilized, giving sodium3-hydroxymethyl-7-hydroxy-7-(2-thienylacetamido)decephalosporanate.

EXAMPLE 653-Carbamoyloxymethyl-7-carbamoyloxy-7-(2-thienylacetamido)decephalosporanicacid

To 0.4 g. of sodium3-hydroxymethyl-7-hydroxy-7(2-thienylacetamido)decephalosporanatesuspended in 10 ml. of acetonitrile cooled to 0° C. is added 0.6 ml. ofchlorosulfonyl isocyanate. The reaction mixture is stirred for 2 hoursand then evaporated to dryness. The residue is taken up in a mixture of20 ml. of ethylacetate and 20 ml. of phosphate buffer and the pHadjusted to 1.6. The mixture is stirred for 3 hours at room temperatureand the pH adjusted to 8 and the aqueous phase separated. The organicphase is extracted with pH 8 phosphate buffer. The combined aqueousphase is taken to pH 2 with hydrochloric acid and extracted twice withethylacetate. The ethylacetate extract is dried over anhydrous sodiumsulfate and evaporated and the residue washed with ether to give3-carbamoyloxymethyl-7-carbamoyloxy-7-(2-thienylacetamido)decephalosporanicacid as a yellow solid.

Following the same procedures and using other acylating agents in placeof thienylacetyl chloride, other 7-acylamido analogs of the products ofExamples 63, 64 and 65 are obtained.

EXAMPLE 66 A. Benzhydryl 7-azido-7-phenoxycephalosporanate

To a solution of 2.3 g. of benzhydryl 7-azido-7-bromocephalosporanate in10 ml. of benzene is added 6 g. of phenol followed by 0.82 g. of drysilver fluoroborate and 0.33 ml. of dry pyridine. The mixture is stirredat 23° C. for 6 hours, then filtered, and the residue washed withbenzene. The combined filtrate and washings is washed with pH 2phosphoric acid buffer and with aqueous sodium bicarbonate and thendried over sodium sulfate and evaporated. The residue is placed on ahigh vacuum pump overnight to remove excess phenol, then chromatographedon 100 g. of silica gel. Elution with methylene chloride givesbenzhydryl 7-azido-7-phenoxycephalosporanate as a viscous oil.

B. Benzhydryl 7amino-7-phenoxycephalosporanate

A solution of 3 g. of benzhydryl 7-azido-7-phenoxycephalosporanate in300 ml. of dry dioxane is hydrogenated in the presence of 3 g. ofplatinum oxide at room temperature and atmospheric pressure for 1 hour.Fresh catalyst (3 g.) is added and the hydrogenation continued for 2hours. The dioxane is removed under reduced pressure and the residue istaken up in chloroform and filtered through 20 g. of silica gel packedin a fritted glass funnel. The cake is washed with 500 ml. of chloroformand the combined washings and filtrate are evaporated, leavingbenzhydryl 7-amino-7-phenoxycephalosporanate.

C. Benzhydryl 7-phenoxy-7-(5-thiazolylacetamido)cephalosporanate

A solution of 2.3 g. of benzhydryl 7-amino-7-phenoxycephalosporanate in25 ml. of methanol is cooled in an ice bath and to it is added withmagnetic stirring 1.8 ml. of dry pyridine followed immediately by asolution of 1.5 g. of 5-thiazolylacetyl chloride in 4 ml. of methylenechloride. The mixture is stirred at 2° C. for 15 minutes, then extractedwith pH 2 phosphoric acid buffer and water. The organic phase is driedover anhydrous sodium sulfate and evaporated. The residue ischromatographed on 100 g. of silica gel. Elution with chloroform givesbenzhydryl 7-phenoxy-7-(5-thiazolylacetamido)cephalosporanate.

D. 7-Phenoxy-7-(5-thiazolylacetamido)cephalosporanic acid

A solution of 2 g. of benzhydryl7-phenoxy-7-(5-thiazolylacetamido)cephalosporanate in 8 ml. of anisoleand 16 ml. of trifluoroacetic acid is stirred at room temperature for 5minutes. The excess anisole and trifluoroacetic acid are rapidly removedunder high vacuum and the residue taken up in 50 ml. of methylenechloride and extracted twice with sodium bicarbonate solution. Theaqueous extract is cooled and overlayered with ethylacetate and the pHadjusted to 2.1 with dilute sulfuric acid. The ethylacetate layer isdried over sodium sulfate and evaporated, leaving7-phenoxy-7-(5-thiazolylacetamido)cephalosporanic acid.

EXAMPLE 67 A. Benzhydryl7-chloro-7-(t-butoxycarbonylthio)cephalosporanate

t-Butoxycarbonylsulfenyl chloride is prepared by the addition oft-butanol to chlorocarbonylsulfenyl chloride in 1:1 molar ratio at 30°C. 10 MMoles of t-butoxycarbonylsulfenyl chloride is dissolved in 50 ml.CH₂ Cl₂ and added dropwise to a solution of 10 mmoles of benzhydryl7-diazocephalosporanate in 50 ml. CH₂ Cl₂ cooled to -40° C. in a dry icebath. The addition is carried out over 15 minutes and at the end thetemperature is allowed to rise to -10° C. to -5° C. gradually. Saturatedsodium bicarbonate is added and the organic layer separated and washedwith water. After drying over sodium sulfate the solvent is removed invacuo. The crude product is evaluated by IR (loss of diazo 2100 cm⁻¹,presence of β-lactam 1790 cm⁻¹, presence of ester bands 1745 cm⁻¹). Itis purified further by TLC to give benzhydryl7-chloro-7-(t-butoxycarbonylthio)cephalosporanate in purer form.

B. Benzhydryl 7-azido-7-(t-butoxycarbonylthio)cephalosporanate

To a solution of 5 mmoles lithium azide in 5 ml. of DMF is added 5mmoles benzhydryl 7-chloro-7-(t-butoxycarbonylthio)cephalosporanate. Thesolution is heated at 40° C. to 70° C. for from 3-6 minutes, thenquenched into ice water. The DMF-water solution is extracted with 2×25ml. CHCl₃ washed with saturated sodium bicarbonate solution, 2×50 ml. H₂O and the organic layer is dried over Na₂ SO₄. Evaporation of thesolvent gives the crude product as a mixture of isomers which can bepurified further by conventional chromatography on silica gel. The twoisomers are evaluated by IR (presence of β-lactam 1790 cm⁻¹, presence ofazide band 2100 cm⁻¹, ester bands 1745 cm⁻¹).

C. Benzhydryl7-(2-thienylacetamido)-7-(t-butoxycarbonylthio)cephalosporanate

The mixture of isomers of 7-azido compound prepared above (5 mmoles) isreduced with 3 g. Bolhoffer catalyst (10% Pd/C) at 40 psi at roomtemperature in 50 ml. ethylacetate in the presence of 5 mmoles ofpyridine and 5 mmoles of thienylacetic anhydride. At the end of 1 hourthe catalyst is filtered and the ethylacetate extracted with 2×20 ml. 1N HCl and 2×50 ml. with 10% sodium bicarbonate, 2×50 ml. H₂ O. Theethylacetate is dried over Na₂ SO₄ and the solvent removed in vacuo. Thecrude product, a mixture of isomers, is evaluated by IR (presence ofβ-lactam 1790 cm⁻¹, loss of azide 2100 cm⁻¹, appearance of new amidebands 1680 cm⁻¹).

D. 7-Thienylacetamido-7-mercaptocephalosporanic acid

5 Mmoles thienylacetamido benzhydryl ester prepared above is dissolvedin 10 ml. of anisole and cooled to 0° C. To this is added 20 ml. oftrifluoroacetic acid. The solution is kept at 0° C. and stirred for 1-3hours. The excess trifluoroacetic acid and anisole are removed byevaporation with a vacuum pump. The product is evaluated by IR(appearance of carboxyl 1710 cm⁻¹, β-lactam 1790 cm⁻¹ loss of esterband). The compound absorbs in the UV spectrum λmax. 260-265 ε (8000).

EXAMPLE 68 A. Benzhydryl 7-chloro-7-(carbamoylthio)cephalosporanate

Carbamoylsulfenyl chloride (10 mmoles) formed by the action of 20 mmoleof ammonia on chlorocarbonylsulfenyl chloride at -70° C. in 50 ml. CH₂Cl₂ is added dropwise to a solution of 10 mmoles of benzhydryl7-diazocephalosporanate at -40° C. At the end of the addition thetemperature is permitted to rise slowly to -5° C. Saturated sodiumbicarbonate solution (50 ml.) is added and the organic layer separated,washed with water and dried over sodium sulfate. The solvent is removedin vacuo to yield a gum. The crude product is evaluated by IR (absenceof diazo 2100 cm⁻¹, presence of β-lactam, new carbamoyl absorption 1680cm⁻¹). The crude product can be purified by preparative TLC.

B. Benzhydryl 7-azido-7-(carbamoylthio)cephalosporanate

The reaction is carried out in the same manner as in the preparation ofbenzhydryl 7-azido-7-(t-butoxycarbonylthio)cephalosporanate. The crudeproduct, a mixture of isomers, is evaluated by IR (new azide band 2100cm⁻¹, β-lactam 1790 cm⁻¹ and carbamoyl group 1680 cm⁻¹).

C. Benzhydryl 7-thienylacetamido-7-(carbamoylthio)cephalosporanate

Reduction-acylation is carried out in the same manner as that forbenzhydryl 7-azido-7-(t-butoxycarbonylthio)cephalosporanate. The crudeproduct is evaluated by IR (loss of azide 2100 cm⁻¹, presence ofβ-lactam, new amide at 1680 cm⁻¹). It may be purified further bypreparative TLC or column chromatography.

D. 7-Thienylacetamido-7-(carbamoylthio)cephalosporanic acid

Removal of the benzhydryl ester is accomplished in the same manner asfor the 7-thienylacetamido-7-butoxycarbonylthiocephalosporanate. Thecrude product is evaluated by IR (ester band disappears at 1740 cm⁻¹,acid band appears at 1710 cm⁻¹) and UV spectrum λmax. 260-265 ε (8000).

EXAMPLE 69 A. Benzhydryl 7-bromo-7-methylthiocephalosporanate

10 Mmoles of benzhydryl 7-diazocephalosporanate dissolved in 100 ml.methylene chloride is cooled to -40° C. under N₂. To this is added 12mmoles methylsulfenyl bromide in 100 ml. CH₂ Cl₂ dropwise with vigorousstirring. Nitrogen evolution is immediate. After addition of the reagent(15 minutes) at -40° C. the mixture is allowed to warm gradually to -5°C. Saturated sodium bicarbonate solution is added and the organic layerseparated and washed with water. After drying over sodium sulfate thesolvent is removed at room temperature in vacuo. The crude product isevaluated by IR (Loss of diazo 2100 cm⁻¹, presence of β-lactam 1790cm⁻¹) and positive Beilstein test for halogen. The crude product can befurther purified by preparative TLC or column chromatography.

B. Benzhydryl 7-azido-7-methylthiocephalosporanate

10 Mmoles of the 7-bromo-7-methylthio compound are heated for 4 minutesat 68° C. in 60 ml. DMF which contains 10 mmoles lithium azide. Thesolution is diluted with 300 ml. water and extracted 2×50 ml.chloroform. The chloroform layer is washed 3×100 ml. water and driedover anhydrous sodium sulfate. The crude product is evaluated by IR(azide 2100 cm⁻¹, β-lactam 1790 cm⁻¹) and negative Beilstein test. Thecrude product is further purified by preparative TLC or columnchromatography.

C. Benzhydryl 7-(2-thienylacetamido)-7-methylthiocephalosporanate

10 Mmoles of 7-azido-7-methylthio compound are dissolved in 50 ml.ethylacetate and 10 mmoles of thienylacetic anhydride, 0.1 ml. pyridineand 800 mg. Bolhoffer catalyst are added. The mixture is hydrogenated atroom temperature for 1 hour. The catalyst is removed by filtration andthe residue evaporated to a glass in vacuo. Infrared analysis of thecrude product shows no azide left 2100 cm⁻¹, appearance of a new amide1680 cm⁻¹ and β-lactam 1790 cm⁻¹. The crude product can be purifiedfurther by preparative TLC or column chromatography.

D. 7-Thienylacetamido-7-methylthiocephalosporanic acid

1 Mmole of the 7-(2-thienylacetamido)-7-methylthio compound (mixture ofisomers) is dissolved in 10 ml. of anisole and cooled to 0° C. To thesolution is added 15 ml. trifluoroacetic acid cooled to 0° C. and themixture is aged at room temperature for 1 hour. The excesstrifluoroacetic acid and anisole are removed by evaporation in vacuo andthe residue flushed twice with chloroform and evaporated to dryness. Thecrude product is evaluated by IR (loss of ester at 1740 cm⁻¹ and theappearance of carboxyl at 1710 cm⁻¹) and UV λmax. 260-262 ε (8000).

EXAMPLE 70 7-(2-Thienylacetamido)-7-acetylthiocephalosporanic acid

10 Mmoles of 7-(2-thienylacetamido)-7-mercapto compound are dissolved in50 ml. pyridine at 0° C. and 10 mmoles of acetyl chloride added dropwiseover 5 minutes. The mixture is quenched in ice water and the pH adjustedto 8 with sodium hydroxide. The pyridine is removed by extraction withether and the aqueous layer lyophilized. The crude product is evaluatedby IR (new carbonyl 1740 cm⁻¹, β-lactam 1790 cm⁻¹) and UV spectrum λmax.260-265 ε (8000).

EXAMPLE 71 7-(2-Thienylacetamido)-7-methylsulfinylcephalosporanic acid

10 Mmoles of the 7-(2-thienylacetamido)-7-Methylthio compound isdissolved in 50 ml. tetrahydrofuran at 0° C. and treated with 10 mmolesof peracetic acid. The solution is stirred at 0° C. for 30 minutes.Sodium thiosulfate solution is added until a negative test with KIpaper. 100 ml. of saturated sodium chloride is added and the organiclayer separated and dried over sodium sulfate. The solvent is removed invacuo and the product evaluated by IR analysis (β-lactam 1790, sulfinylbands 1060 cm⁻¹, 1150 cm⁻¹) and UV spectrum λmax. 260-265.

EXAMPLE 72 Sodium3-hydroxymethyl-7-acetamido-7-methoxydecephalosporanate

Treatment of 7-acetamido-7-methoxycephalosporanic acid in aqueoussolution at pH 6 with acetylesterase obtained from orange peels resultsin the formation of3-hydroxymethyl-7-acetamido-7-methoxydecephalosporanic acid which isrecovered as the sodium salt in accordance with procedures known in thisart.

EXAMPLE 73

Following the procedure described in Example 72 above with other 7-oxyand 7-thio substituted cephalosporins prepared as described in theforegoing examples, the following 3-hydroxymethyldecephalosporanic acidsare prepared:

Sodium3-hydroxymethyl-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Sodium 3-hydroxymethyl-7-methoxy-7-(2-thianaphthene)decephalosporanate

Sodium3-hydroxymethyl-7-methoxy-7-(p-guanidinophenylacetamido)decephalosporanate

Sodium 3-hydroxymethyl-7-methoxy-7-(2-furylacetamido)decephalosporanate

Sodium3-hydroxymethyl-7-methoxy-7-tetraazolylacetamidodecephalosporanate

Sodium3-hydroxymethyl-7-methoxy-7-(D-α-amino-2-phenylacetamido)decephalosporanate

Sodium3-hydroxymethyl-7-methoxy-7-(2-carboxy-2-phenylacetamido)decephalosporanat

Sodium 3-hydroxymethyl-7-benzyloxy-7-phenylacetamidodecephalosporanate

Sodium 3-hydroxymethyl-7-ethoxy-7-(2-thienylacetamido)decephalosporanate

Sodium 3-hydroxymethyl-7-methylthio-7-phenylacetamidodecephalosporanate

Sodium3-hydroxymethyl-7-mercapto-7-(2-thienylacetamido)decephalosporanate

Sodium3-hydroxymethyl-7-carbamoylthio-7-(2-thienylacetamido)decephalosporanate

Sodium3-hydroxymethyl-7-(2-thienylacetamido)-7-(aminocarbonyloxy)decephalosporanate

Sodium3-hydroxymethyl-7-(2-thienylacetamido)-7-(methoxycarbonyloxy)decephalosporanate

Sodium3-hydroxymethyl-7-(2-thienylacetamido)-7-(aminosulfonyloxy)decephalosporanate

EXAMPLE 74 Sodium3-(N,N-dimethylcarbamoyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate

An excess of phosgene is bubbled into a stirred solution of 0.5 g. of3-hydroxymethyl-7-methoxy-7-(2-thienylacetamido)cephalosporanic acid in100 ml. of methylene chloride and the resulting mixture is allowed tostand overnight at room temperature. The excess phosgene is removed bybubbling dry nitrogen through the solution for 3 hours and the resultingsolution is evaporated under reduced pressure.

To a solution of 0.45 g. of the residue in 50 ml. of methylene chloridecooled in an ice bath is added 0.2 g. of dimethylamine. The mixture isstirred for an hour at room temperature and the excess aminehydrochloride is removed by filtration. To the resulting filtrate isadded a solution of 0.1 g. of sodium bicarbonate in 20 ml. of water. Themixture is stirred for 1 hour at room temperature and the aqueous layeris separated, washed twice with methylene chloride, and then freezedried to afford a mixture containing sodium3-(N,N-dimethylcarbamoyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate.

In the same way the other 3-hydroxymethyldecephalosporanic acidcompounds obtained as described in Example 73 are converted to thecorresponding 3-(N,N-dimethylcarbamoyloxymethyl)decephalosporanic acidsalts.

EXAMPLE 75

Following the procedures described in Example 74 using an equivalentamount of piperidine, pyrrolidine or morpholine in place ofdimethylamine, the following products are obtained:

Sodium3-(piperidinocarbonyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Sodium3-(pyrrolidinylcarbonyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Sodium3-(morpholinocarbonyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Sodium3-(piperidinocarbonyloxymethyl)-7-methoxy-7-methylthio-7-(2-thienylacetamido)decephalosporanate

EXAMPLE 76 Sodium3-(N-methylcarbamoyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate

Sodium3-hydroxymethyl-7-methoxy-7-(2-thienylacetamido)decephalosporanate (250mg.) is suspended in dimethylformamide (5 ml.). To this mixture is addedwith agitation 0.2 ml. of trimethylamine and 0.7 ml. ofmethylisocyanate, and the resulting reaction mixture is allowed to standfor 1/2 hour and then evaporated to dryness under reduced pressure. Theresulting residue is taken up in 10 ml. of ethylacetate and 10 ml. of0.1 N phosphate buffer. The pH of the aqueous layer is adjusted to pH1.6 and the mixture stirred for 2 hours at room temperature. The pH isthen adjusted to about 8 with aqueous tripotassium phosphate, and theaqueous phase is separated. The organic phase is reextracted with 10 ml.of pH 8 phosphate buffer. The combined aqueous phase is adjusted to pH2.1 with hydrochloric acid and extracted twice with ethylacetate. Theethylacetate extracts are treated with 10 ml. of water containing 0.15g. of sodium bicarbonate. Separation and freeze drying of the aqueousphase affords sodium3-(N-methylcarbamoyloxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanate.

In the same manner, the 3-hydroxymethylcephalosporanates prepared asdescribed in Example 73 are converted to the corresponding3-(N-methylcarbamoyloxymethyl) compounds.

EXAMPLES 77-86

Following the procedure of Example 76 using other isocyanates, the new3-hydroxymethyl-7-oxy substituted decephalosporanic compounds areconverted to the corresponding N-substituted 3-carbamoyloxymethylcompounds in accordance with the following equation:

    ______________________________________                                         ##STR62##                                                                     ##STR63##                                                                    Example                                                                              OCNR.sub.17        R.sub.17                                            ______________________________________                                        77     OCNCH.sub.2 CH.sub.2 Cl                                                                          CH.sub.2 CH.sub.2 Cl                                78     OCNCH.sub.2 Cl     CH.sub.2 Cl                                         79     OCNC(CH.sub.3).sub.3                                                                             C(CH.sub.3).sub.3                                   80     OCNC.sub.2 H.sub.5 C.sub.2 H.sub.5                                     81     OCNC(CH.sub.3).sub.2 CH.sub.2 Cl                                                                 C(CH.sub.3).sub.2 CHCl                              82     OCNCOOC.sub.2 H.sub.5                                                                            COOC.sub.2 H.sub.5                                  83                                                                                    ##STR64##                                                                                        ##STR65##                                          84                                                                                    ##STR66##                                                                                        ##STR67##                                          85                                                                                    ##STR68##                                                                                        ##STR69##                                          86                                                                                    ##STR70##                                                                                        ##STR71##                                          ______________________________________                                    

wherein R' and R₁ represent the specific substituents shown in Example73.

EXAMPLE 873-Pyridiniummethyl-7-methoxy-7-(2-furylacetamido)decephalosporanic acid

A solution of 1 g. of sodium7-methoxy-7-(2-furylacetamido)cephalosporanate in 5 ml. of water isbrought to pH 2.5 with dilute HCl. Pyridine (8 ml.) is added and thesolution is heated at 70° C. for 31/2 hours. The resulting reactionmixture is lyophilized and the residue is dissolved in water and passedthrough a polystyrene trimethylbenzylammonium anion exchange resin (43%water). The resulting resin adsorbate is eluted with water and selectedfractions are lyophilized to afford substantially pure3-pyridiniummethyl-7-methoxy-7-(2-furylacetamido)decephalosporanic acid.

When an equivalent amount of trimethylamine or triethylamine issubstituted for the pyridine in the foregoing process and otherwisefollowing the described procedure, the corresponding3-(trimethylammoniummethyl) and 3-(triethylammoniummethyl) compounds areobtained.

Similarly, when 3-fluoropyridine, 4-trifluoromethylpyridine,3-carboxypyridine, 4-carbamoylpyridine, 4-(N-methylcarbamoyl)pyridine,4-(N,N-dimethylcarbamoyl)pyridine, 3-(carboxymethyl)pyridine,2-methylpyridine, 3-(hydroxymethyl)pyridine, 3-sulfopyridine or3-cyanopyridine are substituted for the pyridine in the describedprocedure, the corresponding 3- or 4-substituted pyridiniummethylcompounds are obtained.

EXAMPLE 883-Thiouroniummethyl-7-methoxy-7-phenylacetamidodecephalosporanate

A solution of 1 g. of 7-methoxy-7-phenylacetamidocephalosporanic acidand 1 g. of thiourea in 25 ml. of water is maintained at 37° C. for 5days. Acetone (200 ml.) is added and the mixture is cooled in an icebath. The cooled solution is filtered to recover the precipitatedproduct which is then fractionated through a polystyrenetrimethylbenzylammonium anion exchange resin (43% water). Selectedfractions are lyophilized and the crude product is then recrystallizedfrom a mixture of methanol and water to afford3-thiouroniummethyl-7-methoxy-7-phenylacetamidodecephalosporanate.

Upon substituting an equivalent amount of N-methylthiourea,N-ethylthiourea, N,N-dimethylthiourea or N,N-dipropylthiourea in theforegoing process in place of thiourea in the foreoing process andfollowing the procedure described therein, the correspondingN-substituted isothiouronium compounds are obtained.

EXAMPLE 893-(Ethylthiomethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid

A mixture of 7-methoxy-7-(2-thienylacetamido)cephalosporanic acid (0.65g.) and ethanethiol (0.37 ml.) in 10 ml. of 50% aqueous acetone isstirred at room temperature and a 10% aqueous sodium hydroxide solution(2 ml.) is added with stirring. The resulting reaction mixture is thenheated in a sealed glass tube for 100 hours and the resulting mixture isconcentrated under reduced pressure. The residue is dissolved in waterand fractionated through a polystyrene trimethylbenzylammonium anionexchange resin (43% water). Selected fractions are combined andlyophilized to afford3-(ethylthiomethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid.

Upon substituting an equivalent amount of methanethiol, propanethiol,pyridine-2-thiol, pyridine-3-thiol, pyridine-4-thiol,benzothiazole-2-thiol, 4-methylpyrimidine-2-thiol or2-methyl-3,4-thiadiazole-5-thiol for the ethanethiol in the foregoingprocess and otherwise following the described procedure, there is thusobtained the 3-(methylthiolmethyl), 3-(propylthiomethyl),3-(2-pyridylthiomethyl), 3-(3-pyridylthiomethyl),3-(4-pyridylthiomethyl), 3-(2-benzothiazolylthiomethyl),3-(4-methylpyrimidin-2-ylthiomethyl), and3-(2-methyl-3,4-thiadiazol-5-ylthiomethyl) substituted7-methoxy-7-(2-thienylacetamido)decephalosporanic acids, respectively.

EXAMPLE 903-(N,N-dimethylthiocarbamoylthiomethyl)-7-methoxy-7-tetraazolylacetamidodecephalosporanicacid

A solution of 7-methoxy-7-tetraazolylacetamidocephalosporanic acid (6.8g.) and sodium N,N-dimethyldithiocarbamate (2.8 g.) in 60 ml. of wateris heated to 50° C. for 24 hours. The product is lyophilized and thenfractionated through a polystyrene trimethylbenzylammonium anionexchange resin (43% water). Selected fractions are then lyophilized toafford3-(N,N-dimethylthiocarbamoylthiomethyl)-7-methoxy-7-tetraazolylacetamidodecephalosporanicacid.

Upon substituting an equivalent amount of the following reactants:sodium N-methyldithiocarbamate, sodium N,N-diethyldithiocarbamate,sodium N,N-di-n-propyldithiocarbamate, sodiumN-methyl-N-(2-dimethylaminoethyl)-dithiocarbamate, sodiumN-ethyl-n-(2-diethylaminoethyl)dithiocarbamate, sodiumN-(2-di-n-propylaminoethyl)dithiocarbamate, sodiumN-methyl-N-(2-morpholinoethyl)eithiocarbamate, sodiumN-methyl-N-(3-diethylaminopropyl)dithiocarbamate, sodiumN-phenyl-N-(2-methylaminoethyl)dithiocarbamate, sodiumN,N-tetramethylenedithiocarbamate, sodiumN,N-pentamethylenedithiocarbamate, sodiumN,N-bis-(2-hydroxyethyl)dithiocarbamate or the sodium salt of4-methyl-piperazinodithiocarboxylate for the sodiumdimethyldithiocarbamate in the foregoing process, but otherwisefollowing the described procedure, there is thus obtained thecorresponding 3-(N-methylthiocarbamoylthiomethyl),3-(N,N-diethylthiocarbamoylthiomethyl),3-(N,N-di-n-propylthiocarbamoylthiomethyl),3-[N-methyl-N-(2-dimethylaminoethyl)thiocarbamoylthiomethyl],3-[N-ethyl-N-(2-diethylaminoethyl)thiocarbamoylthiomethyl],3-[N-(2-di-n-propylaminoethyl)thiocarbamoylthiomethyl],3-[N-methyl-N-(2-morpholinoethyl)thiocarbamoylthiomethyl],3-[N-methyl-N-(3-diethylaminopropyl)thiocarbamoylthiomethyl],3-[N-phenyl-N-(2-methylaminoethyl)thiocarbamoylthiomethyl],3-(N,N-tetramethylene)thiocarbamoylthiomethyl,3-(N,N-pentamethylene)thiocarbamoylthiomethyl,3-[N,N-bis-(2-hydroxyethyl)thiocarbamoylthiomethyl], and3-(4-methylpiperazino)thiocarbamoylthiomethyl7-methoxy-7-tetraazolylacetamidodecephalosporanic acids.

In the same way other 7-substituted cephalosporins produced as describedin the foregoing examples are converted to the corresponding3-substituted compounds as described above.

EXAMPLE 913-(Benzoylthiomethyl)-7-methoxy-7-(2-carboxy-3-phenylacetamido)decephalosporanicacid

A mixture of 7-methoxy-7-(2-carboxy-2-phenylacetamido)cephalosporanicacid (0.654 g.), sodium bicarbonate (0.504 g.) and thiobenzoic acid(0.414 g.) in 5.0 ml. of water is heated at 50° C. overnight under anitrogen atmosphere. The product is precipitated by the addition ofacetone and crystallized from a mixture of alcohol and water to afford3-(benzoylthiomethyl)-7-methoxy-7-(2-carboxy-3-phenylacetamido)decephalosporanicacid.

Upon substituting an equivalent amount of potassium ethyl xanthate,potassium n-propyl xanthate, potassium isopropyl xanthate, potassiumn-butyl xanthate, potassium n-hexyl xanthate, potassium cyclopentylxanthate and potassium cyclohexyl xanthate for the thiobenzoic acid inthe foregoing process and otherwise following the described procedure,the corresponding 3-(ethoxythiocarbonylthiomethyl),3-(n-propoxythiocarbonylthiomethyl),3-(isopropoxythiocarbonylthiomethyl),3-(n-butoxythiocarbonylthiomethyl),3-(n-hexyloxythiocarbonylthiomethyl),3-(cyclopentyloxythiocarbonylthiomethyl), and3-(cyclohexyloxythiocarbonylthiomethyl)7-methoxy-7-(2-carboxy-2-phenylacetamido)decephalosporanic acids areobtained.

In like manner, the other 7-substituted cephalosporins produced asdescribed in the foregoing examples can be converted to thecorresponding 3-substituted compounds.

EXAMPLE 923-(Toluene-p-sulfonylmethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid

A mixture of 7-methoxy-7-(2-thienylacetamido)cephalosporanic acid (0.654g.) and sodium toluene-p-sulfinate (1.0 g.) in 5.0 ml. of water isheated at 50° C. for 24 hours. The mixture is concentrated in vacuo andcrystallized from a mixture of methanol and water to afford3-(toluene-p-sulfonylmethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid.

EXAMPLE 933-(Azidomethyl)-7-methoxy-7-(2-furylacetamido)decephalosporanic acid

A mixture of 7-methoxy-7-(2-furylacetamido)cephalosporanic acid (2.0 g.)and sodium azide (1.0 g.) are dissolved in 10 ml. water and heated at50° C. overnight. The mixture is then lyophilized to afford crude3-(azidomethyl)-7-methoxy-7-(2-furylacetamido)decephalosporanic acid.

Alternatively, in lieu of treating7-methoxy-7-(2-furylacetamido)cephalosporanic acid with sodium azide, itis possible to substitute3-(carbamoyloxymethyl)-7-(2-furylacetamido)-7-methoxydecephalosporanicacid therefor in an otherwise analogous process to afford an identicalproduct. The following example illustrates this method of preparation:

3-(Carbamoyloxymethyl)-7-(2-furylacetamido)-7-methoxydecephalosporanicacid (100 mg.) in a 0.5 M phosphate buffer solution (5 ml., obtained byadding 3.5 g. of sodium dihydrogen phosphate and 3.4 g. of disodiumphosphate in 100 ml. of water followed by the addition of sufficienthydrochloric acid to bring the pH to 5) is heated in the presence ofsodium azide (20 mg.) at 95° C. for 8 minutes. Preparative thin layerchromatography gives 20 mg. of3-(azidomethyl)-7-methoxy-7-(2-furylacetamido)decephalosporanic acid asindicated by infrared and nuclear magnetic resonance identification.Treatment of this material with trifluoroacetic acid (1.0 ml.) at 0° C.for 5 minutes followed by quenching in a large volume of ether andevaporation of the solvent affords 15 mg. of3-(azidomethyl)-7-methoxy-7-(2-furylacetamido)decephalosporanic acid.

EXAMPLE 943-(2,4-Dihydroxybenzyl)-7-methylthio-7-phenylacetamidodecephalosporanicacid

A mixture of 7-methylthio-7-phenylacetamidocephalosporanic acid (0.654g.), resorcinol (1.1 g.) and water (10 ml.) are heated at 50° C. for 2days. The reaction mixture is then lyophilized to afford3-(2,4-dihydroxybenzyl)-7-methylthio-7-phenylacetamidodecephalosporanicacid.

EXAMPLE 953-(N-methylindol-3-yl)-7-benzyloxy-7-phenylacetamidodecephalosporanicacid

A solution of N-methylindole (0.655 g.) in acetone (5 ml.) is added to asolution of 7-benzyloxy-7-phenylacetamidocephalosporanic acid (0.654 g.)in water (5 ml.) at 50° C. The mixture is heated for 48 hours and thesolvent is then removed in vacuo and the residue triturated with etherto afford3-(N-methylindol-3-yl)-7-benzyloxy-7-phenylacetamidodecephalosporanicacid.

EXAMPLE 96 3-Methyl-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid

A 10% palladium-on-charcoal catalyst is suspended in water (80 ml.) andtreated with hydrogen. The catalyst is then filtered and suspended againin water (50 ml.) and to this mixture (2.67 g.) is added sodium7-methoxy-7-(2-thienylacetamido)cephalosporanate (1.0 g.) in water (10ml.). The resulting mixture is shaken for 22 hours at room temperature.

The catalyst is removed by filtration and washed once with water (50ml.). The combined wash and filtrate is then concentrated to dryness toafford 3-methyl-7-methoxy-7-(2-thienylacetamido)decephalosporanic acid.

EXAMPLE 973-(Amidinothiomethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid

7-Methoxy-7-(2-thienylacetamido)cephalosporanic acid (100 mg.) is heatedfor 8 minutes with thiourea (26 mg.) at 95° C. in a 0.5 M phosphatebuffer solution (5 ml., obtained by adding 3.5 g. of sodium dihydrogenphosphate and 3.4 g. of disodium phosphate in 100 ml. of water followedby the addition of sufficient hydrochloric acid to bring the pH to 5).Electrophoresis of the solution at pH 7 shows the thiouronium compoundas a non-mobile entity. The mixture is purified by absorption on apolystyrene nuclear sulfonic acid cation exchange resin on the hydrogencycle (Dowex 50) to remove the phosphate buffer. Elution is carried outusing a 0.1 N pyridine solution. The pH is adjusted to 8 with 1 N sodiumhydroxide and evaporated under vacuum to remove residual pyridine.Lyophilization gives the title compound.

EXAMPLE 983-(4-Methylthiazol-2-ylmercaptomethyl)-7-methylthio-7-(2-thienylacetamido)decephalosporanicacid

7-Methylthio-7-(2-thienylacetamido)cephalosporanic acid (100 mg.) in 5ml. of a pH 7 buffer (a 0.5 M solution of a mixture of 3.5 g. sodiumdihydrogen phosphate and 3.4 g. of disodium phosphate in 100 ml. ofwater) containing 2-mercapto-4-methylthiazole (50 mg.) is heated at 95°C. for 8 minutes. The resulting reaction mixture contains the titlecompound.

EXAMPLE 993-(1,3,4-Thiadiazol-2-ylmercaptomethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanicacid

By reacting 2-mercapto-1,3,4-thiadiazole instead of2-mercapto-4-methylthiazole and7-methoxy-7-(2-thienylacetamido)cephalosporanic acid and otherwisefollowing the procedure described in Example 98, there is thus obtainedthe title compound.

EXAMPLE 1003-(Thiocyanatomethyl)-7-methoxy-7-(2-furylacetamido)decephalosporanicacid

7-Methoxy-7-(2-furylacetamido)cephalosporanic acid (100 mg.) is added toa 0.5 M buffer solution (5 ml.) consisting of 3.5 g. sodium dihydrogenphosphate and 3.4 g. disodium phosphate in 100 ml. of water and the pHof the mixture is brought to pH 5 by the addition of hydrochloric acid.Sodium thiocyanate (20 mg.) is added and the mixture is heated at 95° C.for 8 minutes. There is thus obtained the title compound.

EXAMPLE 1013-(Chloromethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanic acidtrifluoroacetate

The benzhydryl ester of 7-methoxy-7-(2-thienylacetamido)cephalosporanicacid (1 mmole) is dissolved in methylene chloride (5 ml.) and themixture is cooled to 0° C. Collidine (1 mmole) is added, followed by thedropwise addition of a solution of phosphorous pentachloride (0.6 mmole)in methylene chloride (5 ml.). The mixture is then stirred for an hourin an ice bath at 0° C. and the resulting solution is extracted withsodium bicarbonate, dilute hydrochloric acid and a saturated solution ofsodium chloride. The mixture is evaporated to dryness and then isolatedby chromatography on a cooled silica gel column using chloroform as theeluant. The product thus obtained is the benzhydryl ester of3-(chloromethyl)-7-(2-thienylacetamido)decephalosporanic acid.

A solution of this benzhydryl ester (1 mmole) in anisole (13 ml.) ispoured into 6.5 ml. of cold (0° C.) trifluoroacetic acid with stirring.After 5 minutes the solution is poured, with stirring, into an ethersolution (1800 ml.) maintained at 0° C. The solid precipitate whichresults is then collected and dried to afford the title compound.

EXAMPLE 102 Sodium3-[N-(2-chloroethyl)carbamoyloxymethyl]-7-methoxy-7-(2-thienylacetamido)decephalosporanate

A suspension of 100 mg. of3-hydroxymethyl-7-(2-thienylacetamido)decephalosporanic acid potassiumsalt in 3 ml. dry DMF is placed under N₂ and agitated by ultrasonicwaves. 0.1 Ml. of triethylamine and 0.16 ml. β-chloroethylisocyanate areadded. After 2 hours the solution is diluted with ethyl ether andcentrifuged. The ether is decanted and the oily residue washed with moreether. After recentrifuging the ether is again decanted off. The solidresidue is dissolved in water and the pH adjusted to 2 with concentratedHCl. The product is extracted with ethylacetate which has been washedwith 5% NaHCO₃ solution. The ethylacetate is dried with MgSO₄, filteredand evaporated. The residue is redissolved in ethylacetate and washedwith an aqueous solution of 16 mg. NaHCO₃, the pH of the aqueoussolution is adjusted to 7.6 with NaHCO₃ and the mixture is stirred for1/2 hour. The layers are separated and the water solution washed withEtOAc and then freeze dried overnight.

The 61 mg. of crude material so obtained is dissolved in methanol andall insoluble material filtered out. The methanol is evaporated and asmall amount of ethyl ether added to start solidification. Yield: 55mg., one spot on TLC, Rf of 0.54 in butanol:ethanol:water (4:1:5) upperlayer.

EXAMPLE 103 Disodium3-[N-(4-sulfophenyl)carbamoyloxymethyl]-7-methoxy-7-(2-thienylacetamido)decephalosporanate

A suspension of 200 mg. of3-(hydroxymethyl)-7-methoxy-7-(2-thienylacetamido)decephalosporanic acidpotassium salt in 5 ml. dry DMF is placed under N₂ and agitated byultrasonic waves. 0.096 ml. of triethylamine is added and then 94.4 mg.p-chlorosulfonylphenylisocyanate in 1 ml. DMF is added. After 5 minutesthe solution is diluted with ethyl ether and centrifuged. The ether isdecanted and the green solid residue is washed with more ether. Afterrecentrifuging the ether is again decanted off. The remaining solid isdissolved in 30 ml. of water containing 50 mg. NaHCO₃. After stirringfor 1/2 hour the pH is adjusted to 7.1 with dilute HCl. The solution isfreeze dried overnight to give 295 mg. crude material which is dissolvedin warm MeOH and filtered from insoluble material. A small amount ofisopropanol is added and the first precipitate is filtered off anddiscarded. More isopropanol is then added and two crops of good materialobtained in 39 mg. and 74 mg. yield of the title compound.Electrophoresis in 10% acetic acid shows a single spot.

EXAMPLE 104 A. Benzhydryl 7β-bromo-7-α-methoxycephalosporanate

To a solution of 1.8 g. of benzhydryl 7-diazocephalosporanate in 20 ml.of methylene chloride is added a cold solution of 560 mg. ofN-bromoacetamide in 20 ml. of methanol. The mixture is stirred at roomtemperature for 30 minutes and the solvents are rapidly removed underwaterpump vacuum. The gummy residue is taken up in methylene chlorideand washed with sodium bicarbonate solution, and the organic phase isdried over anhydrous magnesium sulfate and evaporated. The residual oilis chromatographed in 60 g. of silica gel. Elution with methylenechloride gives benzhydryl 7-β-bromo-7-α-methoxycephalosporanate.

B. Benzhydryl 7-α-azido-7-β-methoxycephalosporanate

A solution of benzhydryl 7-β-bromo-7-α-methoxycephalosporanate (700 mg.)and lithium azide (435 mg.) in 5 ml. of dimethyl formamide is stirred at30°-35° C. for 6 hours. The DMF is evaporated under high vacuum and theresidue is taken up in methylene chloride and extracted four times withwater. The methylene chloride phase is dried over anhydrous sodiumsulfate and evaporated. The residual oil is chromatographed in 20 g. ofsilica gel. Elution with hexanemethylene chloride gives benzhydryl7-α-azido-7-β-methoxycephalosporanate.

C. Benzhydryl 7-α-amino-7-β-methoxycephalosporanate

Benzhydryl 7-α-azido-7-β-methoxycephalosporanate (2 g.) and platinumoxide (2 g.) in 200 ml. of dioxane are vigorously stirred under anatmosphere of hydrogen for 2 hours. The dioxane is evaporated underreduced pressure and the residue is taken up in chloroform and filteredthrough 20 g. of silica gel packed in a fritted glass funnel. The cakeis washed with 500 ml. of chloroform and the combined filtrate andwashings are evaporated, leaving benzhydryl7-α-amino-7-β-methoxycephalosporanate.

D. Benzhydryl 7-β-methoxy-7-α-(2-thienylacetamido)cephalosporanate

To an ice-cooled solution of 0.9 g. of benzhydryl7-α-amino-7-β-methoxycephalosporanate in 20 ml. of methylene chloride isadded 0.5 ml. of dry pyridine followed by 0.15 ml. of thienylacetylchloride in 5 ml. of cold methylene chloride. The mixture is stirred inan ice-bath for 10 minutes, then washed successively with pH 2phosphoric acid buffer, water and sodium bicarbonate solution. Themethylene chloride phase is dried over anhydrous sodium sulfate andevaporated and the residue is chromatographed on 20 g. of silica gel.Elution with chloroform yields benzhydryl7-β-methoxy-7-α-(2-thienylacetamido)cephalosporanate.

E. Sodium 7-β-methoxy-7-α-(2-thienylacetamido)cephalosporanate

A solution of 0.8 g. of benzhydryl7-β-methoxy-7-α-(2-thienylacetamido)cephalosporanate in 3 ml. of anisoleand 7 ml. of trifluoroacetic acid is stirred at room temperature for 5minutes and the excess anisole and trifluoroacetic acid are then rapidlypumped off on a vacuum pump. The residue is taken up in a mixture ofmethylene chloride and water and rapidly stirred while sodiumbicarbonate is added to bring the pH to 6.5. The aqueous layer isseparated, washed with ether and lyophilized, affording sodium7-β-methoxy-7-α-(2-thienylacetamido)cephalosporanate.

F. Epimerization of benzhydryl 7-β-bromo-7-α-methoxycephalosporanate

A solution of 250 mg. of benzhydryl7-β-bromo-7-α-methoxycephalosporanate and 0.2 g. of lithium bromide in1.5 ml. of dimethylformamide is stirred at room temperature overnight.The DMF is removed on the vacuum pump and the residue is taken up inchloroform and washed with water. The chloroform is evaporated, leavingan equilibrium mixture containing predominantly benzhydryl7-α-bromo-7-β-methoxycephalosporanate. This is separated from thestarting epimer by chromatography on silica gel.

G. Benzhydryl 7-β-azido-7-α-methoxycephalosporanate

This is prepared as in B starting with benzhydrylα-bromo-7-β-methoxycephalosporanate. The product is identical in allrespects with benzhydryl 7-β-azido-7-α-methoxycephalosporanatepreviously prepared by the action of silver fluoroborate in methanol onbenzhydryl 7-azido-7-bromocephalosporanate.

EXAMPLE 105 Pivaloyloxymethyl3-carbamoyloxy-7-α-methoxy-7-(2-thienylacetamido)decephalosporanate

A mixture of 0.90 g. of sodium3-carbamoyloxy-7-α-methoxy-7-(2-thienylacetamido)decephalosporanate,0.30 g. of chloromethyl pivalate, and 5 ml. of dry dimethylformamide isshaken overnight at room temperature. The reaction mixture is thenpoured onto 25 ml. of pH 6 phosphate buffer and extracted with 3×10 ml.of chloroform. The combined extracts are washed with 4×10 ml. of waterand 10 ml. of saturated brine, and dried over anhydrous magnesiumsulfate. Evaporation of the solvent in vacuo leaves a gum which iswashed by decantation with petroleum ether to afford pivaloyloxymethyl3-carbamoyloxy-7-α-methoxy-7-(2-thienylacetamido)decephalosporanate.

EXAMPLE 106 A. Benzhydryl7-(p-nitrobenzylideneamino)-7-trifluoromethoxycephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate (571 mg.) isstirred at 0° C. under nitrogen in 10 ml. of acetonitrile.Bis(trifluoromethyl)peroxide (170 mg.) is added, and then over a 1 hourperiod 387 mg. diisopropylethylamine in 5 ml. acetonitrile.

The reaction mixture is evaporated in vacuo, taken up in 25 ml. benzene,filtered, and washed successively with water, dilute phosphoric acid(buffered at pH 2), water and aqueous bicarbonate. The solution is driedwith MgSO₄, filtered and evaporated, providing benzhydryl7-(p-nitrobenzylideneamino)-7-trifluoromethoxycephalosporanate as amixture of epimers at C-7. These are separated by chromatography onsilica gel, eluting with 4:1 chloroform-ethylacetate.

B. Benzhydryl 7-β-amino-7-α-trifluoromethoxycephalosporanatehydrochloride

The 7β-(p-nitrobenzylideneamino)-7β-trifluoromethoxy isomer (655 mg.)and 260 mg. aniline hydrochloride are stirred together for 5 hours at25° C. in 10 ml. methanol. The methanol is removed at 0.1 mm. pressureand 30° C., and the residue covered with ether to crystallize for 1hour. The solid is triturated with ether, filtered, and washed withether several times. It consists of the title compound and anilinehydrochloride mixed together, and is used immediately for the next step.

The other epimer is converted to the epimeric compound in the samemanner.

C. Benzhydryl7β-(2-thienylacetamido)-7α-trifluoromethoxycephalosporanate

The mixture of product and aniline hydrochloride obtained as describedin B above is stirred vigorously at -10° C. in 25 ml. methylenechloride. Thienylacetyl chloride (0.5 g.) is added, and then 0.5 g.triethylamine is slowly introduced. This liberates the free amine,benzhydryl 7β-amino-7α-trifluoromethoxycephalosporanate, which isinstantly acylated. The reaction mixture is slowly allowed to warm toroom temperature. Excess acid chloride is hydrolyzed by shaking withwater, and the methylene chloride layer is then washed successively withdilute phosphoric acid (buffered to pH 2), water, and dilutebicarbonate. After drying with MgSO₄, the solution is filtered andevaporated, affording a mixture of the product and N-phenylthienylacetamide. Pure product is obtained by silica gel chromatography,eluting with 4:1 chloroform-ethylacetate.

The other epimer at C-7 is converted to the epimeric compound using thesame procedures.

D. Sodium 7β-(2-thienylacetamido)-7α-trifluoromethoxycephalosporanate

Benzhydryl 7β-(2-thienylacetamido)-7α-trifluoromethoxycephalosporanate(646 mg.) is dissolved in 0.8 ml. anisole and cooled to 0° C.Trifluoroacetic acid (4 ml.) precooled to 0° C. is added and thereaction allowed to proceed for 2 minutes at 0° C. Vacuum of 0.1 mm. isimmediately applied and the reaction mixture allowed to warm to roomtemperature. Anisole is then distilled at 30° C./0.1 mm. A few ml.anisole is added to the residue and pulled off at 30° C./0.1 mm. toinsure total removal of trifluoroacetic acid. The product is treatedwith 5 ml. water containing 84 mg. NaHCO₃ and lyophilized. The resultingpowder is washed thoroughly with ether and dried, affording the titlecompound.

The sodium salt of the other epimer at C-7 is obtained in the samemanner.

EXAMPLE 107 7-(D-α-sulfoaminophenylacetamido)-7-methoxycephalosporanicacid disodium salt

A suspension of 10 mmoles of7-(D-α-aminophenylacetamido)-7-methoxycephalosporanic acid in 100 ml. ofmethylene chloride containing 12 mmoles of triethylamine is stirred andcooled in an ice bath. 12 Mmoles of trimethylamine sulfur trioxide isadded in small portions over 5 minutes. After 1/2 hour the mixture isfiltered and concentrated to 10% of the original volume. The solution isdiluted with an equal volume of acetone and 30 mmoles of sodium2-ethylhexanoate in butanol is added. A solid is formed upon chillingand scratching and is collected by filtration. The disodium salt of7-(D-α-sulfoaminophenylacetamido)-7-methoxycephalosporanic acid can berecrystallized from isopropanol-water if desired.

EXAMPLE 108 Disodium7α-cyano-7(2-carboxyphenylacetamido)cephalosporanate A. Benzhydryl7α-cyano-7-azidocephalosporanate

To a solution of benzhydryl-7-bromo-7-azidocephalosporanate (0.543 g.0.001 mole) in 10 ml of CH₃ CN is added a solution of 0.350 g oftetrabutyl ammonium cyanide in 15 ml of CH₃ CN. The reaction mixture isstirred at room temperature overnight, diluted with CH₂ Cl₂ and washedwith water, dried and evaporated. Chromatography on silica gel gives thebenzhydryl 7α-cyano-7-azidocephalosporanate and the benzhydryl7β-cyano-7-azidocephalosporanate.

The tetrabutyl ammonium cyanide is prepared as follows:

1 G of tetrabutylammonium iodide is dissolved in 10 ml of a 20% aqueousNaCN solution. The aqueous phase is discarded. The organic phase istreated with 3 further 5 ml quantities of the NaCN solution. The organicphase is dried over MgSO₄ and evaporated to give tetrabutylammoniumcyanide.

B. Benzhydryl 7α-cyano-7-aminocephalosporanate

0.500 G of benzhydryl 7α-cyano-7-azidocephalosporanate is dissolved in50 ml of ethyl acetate, 0.500 g of 10% Pd on carbon catalyst is addedand the mixture is stirred under H₂ overnight. The catalyst is filteredoff, the filtrate is evaporated and the residue is chromatographed onsilica gel to separate starting material from product.

C. Benzhydryl7α-cyano-7-(2-benzhydryloxycarbonylphenylacetamido)cephalosporanate

0.473 G of the benzhydryl-7α-cyano-7-aminocephalosporante is dissolvedin 25 ml of CH₂ Cl₂ and cooled to 0° C.2-Benzhydryloxycarbonylphenylacetyl chloride 0.400 g is then addedfollowed 1 minute later with 0.200 g of pyridine. The reaction mixtureis stirred at 0° C. of 25 minutes and poured onto crushed ice. Themixture is agitated and the organic phase is separated and washed oncewith 5% NaHCO₃ solution, once with pH 2 phosphate buffer and once withwater. The organic phase is dried over sodium sulfate and evaporated togive the crude product. Chromatography over silica gen gives thepurified product.

D. Disodium 7α-cyano-7-(2-carboxyphenylacetamido)cephalosporanate

Benzhydryl7α-cyano-7-(2-benzhydryloxycarbonylphenylacetamido)cephalosporanate0.350 g is dissolved in 3 ml of anisole and treated with 10 ml oftrifluoroacetic acid at room temperature for 10 minutes. Thetrifluoroacetic acid and the anisole are removed under reduced pressurebelow 40° C. and the residue is taken up in 25 ml of methylisobutylketone and treated with 0.300 g of NaHCO₃ in 30 ml of H₂ O. The mixtureis stirred for 1/2 hour, the organic phase is separated and the aqueousphase washed twice with methylene chloride and then lyophilized. Thesolid product is purified by crystallization from MeOH/isopropanol.

EXAMPLE 109

A. Benzhydryl 7-azido-7-carboxycephalosporanate

Benzhydryl 7-azido-7-bromocephalosporanate (5.43 g., 0.01 mole) isdissolved in 20 ml. of dry ether and cooled to -20° C., and 10 ml. of 1M phenyl lithium solution is added slowly with vigorous stirring. After1 hour at room temperature the mixture is poured onto pulverized dryice. The residue is extracted with water and acidified to yieldbenzhydryl 7-azido-7-carboxycephalosporanate.

B. Benzhydryl 7-azido-7-chloroformylcephalosporanate

Benzhydryl 7-azido-7-carboxycephalosporanate (4.0 g.) is added to 15 ml.of thionyl chloride and the mixture stirred in an ice bath for 1 hour.The excess thionyl chloride is removed in vacuo and the residue flushedwith dry acetone to give crude benzhydryl7-azido-7-chloroformylcephalosporanate.

C. Benzhydryl 7-azido-7-carbomethoxycephalosporanate

Benzhydryl 7-azido-7-chloroformylcephalosporanate (2.63 g., 0.005 m.) isdissolved in methylene chloride (15 ml.) at 5° C. Pyridine (40 mg.) andmethanol (1.0 ml.) are added slowly. The solvent is removed in vacuo andthe product isolated by chromatography on silica gel.

D. Benzhydryl 7-amino-7-carbomethoxycephalosporanate

1.0 g. of benzhydryl 7-azido-7-carbomethoxycelphalosporanate isdissolved in 100 ml. of dioxane. 1.0 g. of platinum oxide is added andthe reaction mixture stirred under hydrogen at atmospheric pressure for1 hour. Another 1.0 g. quantity of platinum oxide is added, and thereaction mixture is again placed under hydrogen and stirred for 3 hoursuntil the azide is completely reacted as determined by infrared analysisof aliquots. The solvent is removed under reduced pressure and theresidue taken up in 50 ml. of chloroform and filtered through silica gelG in chloroform in a 60 ml. sintered glass funnel. The material iseluted with chloroform until 200 ml. of chloroform has been collected.The chloroform is removed under reduced pressure affording 0.632 g. ofbenzhydryl-7-amino-7-carbomethoxycephalosporanate, which is acylateddirectly without further purification.

E. Benzhydryl 7-carbomethoxy-7-(2-thienylacetamido)-cephalosporanate

0.632 g. of benzhydryl 7-amino-7-carbomethoxycephalosporanate is takenup in 25 ml. of methylene chloride and cooled to 0° C. 0.6 ml. (0.038m.) of 2-thienyl acetyl chloride is added dropwise over 30 secondsfollowed by 0.6 ml. (0.01 m.) of pyridine 60 seconds later. The reactionmixture is stirred at 0° C. for 15 minutes and poured into crushed ice.The mixture is agitated and the organic layer separated and washed oncewith 20 ml. of water, once with 20 ml. of 5% sodium bicarbonate and onceagain with 20 ml. of water. The methylene chloride is dried andevaporated to dryness affording 1.417 g. of crude product. This materialis placed on a column of 60 g. of silica gel under benzene and thecolumn is eluted with benzene taking 100 ml. fractions followed by 300ml. of methylene chloride/benzene (1:1) in 3 fractions, and 500 ml. ofmethylene chloride in 5 fractions. The product is removed from thecolumn by eluting with 400 ml. of chloroform in 4 fractions, affording0.592 g. This material is taken up in 25 ml. of methylene chloride andstirred at room temperature with 20 ml. of solution of 0.120 g. of 0.120g. of sodium bicarbonate in water for 1/2 hour. The layers are separatedand the organic layer washed with water, dried annd evaporated todryness, affording 0.420 g. of benzhydryl7-carbomethoxy-7-(2-thienylacetamido)cephalosporanate.

F. Sodium 7-carbomethoxy-7-(2-thienylacetamido)cephalosporanate

0.420 g. of benzhydryl7-carbomethoxy-7-(2-thienylacetamido)cephalosporanate is dissolved in3.5 ml. of anisole and treated with 10 ml. of trifluoroacetic acid atroom temperature for 10 minutes. The trifluoroacetic acid and anisoleare removed under reduced pressure maintaining the temperature below 40°C., and the residue is taken up in 25 ml. of chloroform and treated with20 ml. of water containing 0.120 g. of sodium bicarbonate. The mixtureis stirred for 1/2 hour at room temperature, and the organic phase isseparated and washd with water. The combined aqueous phase is washedtwice with methylene chloride and lyophilized to afford 0.382 g. ofsodium 7-carbomethoxy-7-(2-thienylacetamido)cephalosporanate as abrownish solid.

EXAMPLE 110

3-Pyridiniummethyl-7-carbomethoxy-7-(2-thienylacetamido)decephalosporanicacid

1.3 g. of sodium 7-carbomethoxy-7-(2-thienylacetamido)cephalosporanateis dissolved in 3 cc. of a buffer solution made up by dissolving 30 g.of potassium iodide in 20 ml. of water containing 5 ml. of pyridine andadjusting the pH to 6.5 with dilute acetic acid. The resulting reactionmixture is stirred at 80° C. for 2 hours and then cooled to roomtemperature. The solution is then freeze dried, dissolved in 40 ml. of25% acetic acid, filtered and the filtered solution subjected toelectrophoresis to obtain3-pyridiniummethyl-7-carbomethoxy-7-(2-thienylacetamido)-decephalosporanicacid.

EXAMPLE 111 Sodium7-dimethylcarboxamido-7-(thiophene-2-acetamido)cephalosporanate

Following the procedure described in Example 107C above, benzhydryl7-azido-7-chloroformylcephalosporanate is reacted with dimethylamine inplace of methanol to produce the corresponding 7-dimethylcarboxamidecompound which is converted to sodium7-dimethylcarboxamido-7-(thiophene-2-acetamido)cephalosporanate usingthe procedures described in Example 109 above.

EXAMPLE 112 Sodium7-hydrazinocarboxyl-7-(2-furylacetamido)cephalosporanate

When benzhydryl 7-azido-7-chloroformylcephalosporanate is reacted withhydrazine in place of methanol using the procedures described in Example109C above, the corresponding 7-hydrazinocarboxycephalosporanate isobtained. This product is reduced to the corresponding 7-amino compoundas described in Example 109D above, and the benzhydryl7-amino-7-hydrazinocarboxylcephalosporanate obtained is reacted withfurylacetyl chloride as described in Example 107E above to obtainbenzhydryl 7-(2-furylacetamido)-7-hydrazinocarboxylcephalosporanate.This product is converted to sodium7-hydrazinocarboxyl-7-(2-furylacetamido)cephalosporanate using theprocedures described in Example 109F above.

EXAMPLE 113 Sodium7-(2-furylacetamido)-7-thiocarboxymethylcephalosporanate

When benzhydryl 7-azido-7-bromocephalosporanate is reacted with carbondisulfide in place of carbon dioxide as described in Example 109A above,benzhydryl 7-azido-7-dithiocarboxycephalosporanate is obtained. Thisproduct is converted to sodium7-(2-furylacetamido)-7-thiocarboxymethylcephalosporanate using theprocedures shown in Examples 107-110 above.

EXAMPLE 114 Sodium 7α-formyl-7-(2-thienylacetamido)cephalosporanate

To a solution of 200 mg. of sodium7α-hydroxymethyl-7-(2-thienylacetamido)cephalosporanate is addedphosphoric acid to a pH of 2-3. The solution is immediately extractedwith ethylacetate in three portions. The combined organic layers arewashed successively with water and saturated sodium chloride solution,and finally dried over anhydrous magnesium sulfate. Concentration of thesolution under reduced pressure at or below room temperature affords7α-hydroxy-7-(2-thienylacetamido)cephalosporanic acid (158 mg.) as a gumwhich hardens to a glass. A solution of this material in 4 ml. of amixture of 1:1 methylene chloride and alcohol-free chloroform is addedat once at room temperature with vigorous agitation to a solutionprepared by the addition of 225 mg. of anhydrous chromium trioxide to amixture of 0.375 ml. of pyridine and 6 ml. of methylene chloride. After5 minutes of agitation the reaction mixture is diluted with 15 ml. ofethylacetate and treated with 4 ml. of 2% hydrochloric acid. The phasesare mixed well and filtered with pressure through diatomaceous earth.The phases are separated and the aqueous layer extracted withethylacetate. The combined organic layers are washed with water anddried over anhydrous magnesium sulfate. Concentration under reducedpressure at room temperature affords 82 mg. of7α-formyl-7-(2-thienylacetamido) cephalosporanic acid as anon-crystalline foam. The solid sodium salt is obtained by dissolvingthe product in one equivalent of aqueous sodium hydroxide followed byremoval of the water by lyophilization.

EXAMPLE 115 Sodium 7α-carboxy-7-(2-thienylacetamido)cephalosporanate

Argentic oxide is prepared by adding 10% potassium hydroxide to anaqueous solution of equimolar quantities of silver nitrate and potassiumpermanganate. The silver (II) oxide which precipitates is recovered byfiltration and washed with water until free of base and permanganate.

To a suspension of 250 mg. of this silver (II) oxide in 1.5 ml. oftetrahydrofuran-water (4:1) is added a solution of 100 mg. of7α-formyl-7-(2-thienylacetamido) cephalosporanic acid in 1.5 ml. oftetrahydrofuran. This mixture is stirred at room temperature until thinlayer chromatographic analysis indicates that the formyl compound iscompletely reacted. The reaction mixture is filtered and the filtratetreated with 2% hydrochloric acid to a pH of 2. Ethylacetate and waterare added to the acidified filtrate and mixed. The separated organiclayer is washed, dried and concentrated under reduced pressure belowroom temperature. The free acid thus obtained is converted to the sodiumsalt as in Example 114.

EXAMPLE 116 Sodium 7-Methyl-7-(2-Thienylacetamido)cephalosporate A.Benzhydryl 7-dimethylboron-7-methylcephalosporanate

A solution of 0.228 g. of benzhydryl 7-diazocephalosporanate in 5 ml. ofmethylene chloride, prepared as described above, is diluted to about 10ml. with tetrahydrofuran and cooled to about -78° C. in a dry ice bath.To this cooled solution is added 3.5 ml. of trimethylboron solution withstirring over a period of 10 minutes. The resulting reaction mixture isallowed to stir for another 10 minutes at -78° C. An infrared spectrumof a small aliquot of this solution at this point shows no diazo band.The reaction mixture containing the benzhydryl7-dimethylboron-7-methylcephalosporanate is allowed to warm up to roomtemperature. Evaporation of the solvent affords the product in solidform.

The trimethylboron solution used in this example is prepared as follows:Methyl magnesium iodide is prepared from 1.44 g. of magnesium and 8.5 g.of methyl iodide in 40 ml. of ether, and the resulting Grignard solutionis diluted to 50 ml. with additional ether. 5 ml. of this solution isplaced in a 3 neck-round bottom flask equipped with dropping funnel,magnetic stirrer and a nitrogen inlet tube. The flask is connected to agas inlet tube fitted with a 2 neck flask containing 5 ml. of ether,cooled to -78° C. in a dry ice bath and both flasks are placed under anitrogen atmosphere; the nitrogen gas being passed into the first flaskand bubbled into the ether in the second flask. 0.2 ml. of borontrifluoride etherate diluted to 2 ml. with ether is added dropwise tothe first flask and the trimethylboron formed is collected in the secondflask. The flow of nitrogen is continued until much of the ether in thefirst flask is flushed into the second flask to give about 9 ml. oftrimethylboron solution.

B. Benzhydryl 7azido-7-methylcephalosporanate

To the methylene chloride solution of benzhydryl7-dimethylboron-7-methylcephalosporanate prepared as described in Aabove is added a solution of bromine azide in methylene chlorideprepared as described below. The resulting reaction mixture turns orangeand is allowed to stir overnight at room temperature. It is then pouredinto cold N/10 sodium thiosulfate solution. The organic phase isseparated, washed with an equal quantity of aqueous 5% sodiumbicarbonate solution and then with water. The washed organic phase isthen dried over sodium sulfate and evaporated to give benzhydryl7-azido-7-methylcephalosporanate.

The solution of bromine azide is prepared as follows:

To a solution of 2.4 gm. of sodium azide in 4 ml. of water is added 40ml. of methylene chloride and the mixture cooled to 0° C. To this cooledmixture is added dropwise 4 ml. of 50% aqueous sulfuric acid (vol./vol.)over 5 minutes and the mixture is allowed to stir another 5 minutes. Theaqueous layer is frozen by cooling in a solid carbon dioxide bath andthe organic phase is decanted off and dried over anhydrous sodiumsulfate. To this solution of the hydrazoic acid is added 2 g. ofN-bromosuccinamide and the mixture is stirred for 15 minutes at 0° C.until all the N-bromosuccinamide is dissolved.

C. Benzhydryl 7-amino-7-methylcephalosporanate

1.0 g. of benzhydryl 7-azido-7-methyl-cephalosporanate is dissolved in100 ml. of dioxane. 1.0 g. of platinum oxide is added and the reactionmixture stirred under hydrogen at atmospheric pressure for 1 hour.Another 1.0 g. quantity of platinum oxide is added, and the reactionmixture is again placed under hydrogen and stirred for 3 hours until theazide is completely reacted as determined by infrared analysis ofaliquots. The solvent is removed under reduced pressure and the residuetaken up in 50 ml. of chloroform and filtered through silica gel G inchloroform in a 60 ml. sintered glass funnel. The material is elutedwith chloroform until 200 ml. of chloroform has been collected. Thechloroform is removed under reduced pressure affording 0.632 g. ofbenzhydryl 7-amino-7-methylcephalosporanate, which is acylated directlywithout further purification.

D. Benzhydryl 7-methyl-7-(2-thienylacetamido)cephalosporanate

0.632 g. of benzhydryl 7-methyl-7-aminocephalosporanate is taken up in25 ml. of methylene chloride and cooled to 0° C. 0.6 ml. (0.038 m.) of2-thienyl acetyl chloride is added dropwise over 30 seconds followed by0.6 ml. (0.01 m.) of pyridine 60 seconds later. The reaction mixture isstirred at 0° C. for 15 minutes and poured into crushed ice. The mixtureis agitated and the organic layer separated and washed once with 20 ml.of water, once with 20 ml. of 5% sodium bicarbonate and once again with20 ml. of water. The methylene chloride is dried and evaporated todryness affording 1.417 g. of crude product. This material is placed ona column of 60 g. of silica gel under benzene and the column is elutedwith benzene taking 100 ml. fractions followed by 300 ml. of methylenechloride/benzene (1:1) in 3 fractions, and 500 ml. of methylene chloridein 5 fractions. The product is removed from the column by eluting with400 ml. of chloroform in 4 fractions, affording 0.592 g. This materialis taken up in 25 ml. of methylene chloride and stirred at roomtemperature with 20 ml. of a solution of 0.120 g. of sodium bicarbonatein water for 1/2 hour. The layers are separated and the organic layerwashed with water, dried and evaporated to dryness, affording 0.420 g.of benzhydryl 7-methyl-7-(2-thienylacetamido)cephalosporanate.

E. Sodium 7-methyl-7-(2-thienylacetamino)cephalosporanate

0.420 g. of benzhydryl 7-methyl-7-(2-thienylacetamido)cephalosporanateis dissolved in 3.5 ml. of anisole and treated with 10 ml. oftrifluoroacetic acid at room temperature for 10 minutes. Thetrifluoroacetic acid and anisole are removed under reduced pressuremaintaining the temperature below 40° C., and the residue is taken up in25 ml. of chloroform and treated with 20 ml. of water, containing 0.120g. of sodium bicarbonate. The mixture is stirred for 1/2 hour at roomtemperature and the organic phase is separated and washed with water.The combined aqueous phase is washed twice with methylene chloride andlyophilized to afford 0.382 g. of sodium7-methyl-7-(2-thienylacetamido)cephalosporanate as a brownish solid.

EXAMPLE 1173-Pyridiniummethyl-7-methyl-7-(2-thienylacetamido)decephalosporanic acid

1.3 g. of sodium 7-methyl-7-(2-thienylacetamido)cephalosporanate isdissolved in 3 cc of a buffer solution made up by dissolving 30 g. ofpotassium iodide in 20 ml. of water containing 5 ml. of pyridine andadjusting the pH to 6.5 with dilute acetic acid. The resulting reactionmixture is stirred at 80° C. for 2 hours and then cooled to roomtemperature. The solution is then freeze dried, dissolved in 40 ml. of25% acetic acid, filtered and the filtered solution subjected toelectrophoresis to obtain3-pyridiniummethyl-7-methyl-7-(2-thienylacetamido)decephalosporanicacid.

EXAMPLE 118 A. Benzhydryl 7-difluoromethylenecephalosporanate

Benzhydryl 7-diazocephalosporanate (4.5 g., 0.01 mole) in 10 ml. of drydioxane is added to a solution of 4.1 g. of 0.05 mole thiocarbonylfluoride and allowed to stand at room temperature until the reddishcolor of the diazo compound disappears. The reaction mixture is heatedat reflux for 45 minutes and the solvent removed in vacuo. The productis chromatographed on silica gel using chloroform: methanol as theeluant to afford benzhydryl 7-difluoromethylenecephalosporanate.

B. Benzhydryl 7-trifluoromethyl-7-bromocephalosporanate

Benzhydryl 7-difluoromethylenecephalosporanate (2.35 g., 0.005 mole) andfinely powdered silver monofluoride (1.90 g., 0.015 mole) in 50 ml. ofbenzene are vigorously stirred. Bromine (0.80 g., 0.005 mole) is addeddropwise. Benzhydryl 7-trifluoromethyl-7-bromocephalosporanate isisolated by chromatography on silica gel.

C. Benzhydryl 7-trifluoromethyl-7-aminocephalosporanate

Benzhydryl 7-trifluoromethyl-7-bromocephalosporanate (1.14 g., 0.002mole) is dissolved in 20 ml. of dry ether and cooled to -20° C. To thiscooled solution is slowly added 0.4 ml. of 5 M ethereal methyllithiumsolution with vigorous stirring. After 1/2 hour, 66 mg. of0-methylhydroxylamine is added. The mixture is kept at -10° C. for 1/2hour and allowed to warm to room temperature before heating at refluxfor 2 hours. The ether is removed in vacuo and the benzhydryl7-trifluoromethyl-7-aminocephalosporanate is isolated by chromatographyon silica gel.

D. Benzhydryl 7-(2-thiopheneacetamido)-7-trifluoromethylcephalosporanate

The benzhydryl 7-trifluoromethyl-7-aminocephalosporanate prepared asdescribed in C above is acylated by reaction with2-thiopheneacetylchloride using the processes described in Example IFabove to afford benzhydryl7-(2-thiopheneacetamido)-7-trifluoromethylcephalosporanate.

E. Sodium 7-(2-thiopheneacetamido)-7-trifluoromethylcephalosporanate

Benzhydryl 7-(2-thiopheneacetamido)-7-trifluoromethylcephalosporanate isdissolved in 3.5 ml. of anisole and treated with 10 ml. oftrifluoroacetic acid at room temperature for 10 minutes. Thetrifluoroacetic acid and anisole are removed under reduced pressuremaintaining the temperature below 40° C., and the residue is taken up in25 ml. of chloroform and treated with 20 ml. of water, containing 0.120g. of sodium bicarbonate. The mixture is stirred for 1/2 hour at roomtemperature and the organic phase is separated and washed with water.The combined aqueous phase is washed twice with methylene chloride andlyophilized affording 0.382 g. of sodium7-(2-thiopheneacetamido)-7-trifluoromethylcephalosporanate.

EXAMPLE 119 A. Benzhydryl 7-ethynyl-7-azidocephalosporanate

A stirring solution of 0.900 g of benzhydryl 7-diazocephalosporanate in10 ml each of methylene chloride and ether is cooled to -78° C. under anitrogen atmosphere and is treated dropwise with a solution oftriethynylboron. The addition is halted periodically, and the progressof the reaction is ascertained by infrared analysis of small aliquots.When the diazo compound has completely reacted, the remainingtriethynylboron solution is discarded. Bromine azide in methylenechloride, 25 ml of a 0.28 N solution, is then added over a period of 20min. The cooling bath is removed and the mixture is stirred for afurther 60 min. at room temperature.

The reaction mixture is poured onto a solution of 20 ml. of 0.1 N sodiumthiosulfate and 20 ml of 0.5 M pH 7 phosphate buffer and agitated. Theorganic phase is separated, washed with water and saturated brine, driedover anhydrous magnesium sulfate, and evaporated in vacuo. The residueis chromatographed on silica gel using benzene as eluant to affordbenzhydryl 7α-ethynyl-7-azidocephalosporanate and benzhydryl7β-ethynyl-7-azidocephalosporanate.

The triethynylboron solution used above is prepared as follows: Asolution of 5.42 g of boron trifluoride in 20 ml of ether is stirred at078° C. under a nitrogen atmosphere while 2.88 g of sodium acetylide in20 ml of xylene is added over a period of 90 min. The reaction mixture,which is a white solid suspended in a clear liquid, is rapidlytransferred to a drybox and filtered into a dry ice jacketed droppingfunnel. This solution is used immediately. At no point is thetemperature of the triethynylboron allowed to exceed -60° C.

B. Benzhydryl 7α-ethynyl-7-aminocephalosporanate

To a stirring solution of 0.028 g of anhydrous cobalt (II) bromide in 20ml of absolute ethanol is added 0.061 g of 2,2'-bipyridine. After allthe bipyridine has dissolved, a solution of 0.315 g of benzhydryl7α-ethynyl-7-azidocephalosporanate in 5 ml of absolute ethanol is addedfollowed by 0.073 g of sodium borohydride. The reaction mixture isstirred for 15 min. at room temperature, and is then quenched by theaddition of cold aqueous acetic acid. The mixture is diluted with 25 mlof water and extracted with three 20 ml portions of ether. The combinedextracts are washed with pH 7 phosphate buffer and saturated brine, anddried over anhydrous magnesium sulfate. Evaporation of the solvent underreduced pressure gives benzhydryl 7α-ethynyl-7-aminocephalosporanate.This material is acylated without further purification.

C. Benzhydryl 7α-ethynyl-7-(2-thienylacetamido)cephalosporanate

An ice cold, stirring solution of 0.274 g of benzhydryl7α-ethynyl-7-aminocephalosporanate in 10 ml of methylene chloride istreated with 0.25 ml of 2-thienylacetyl chloride over a period of 30sec., followed 1 minute later by 0.25 ml of pyridine. The reactionmixture is stirred for a further 15 min. at 0° C., and is then pouredonto cracked ice. The methylene chloride phase is separated, washed with10 ml of water, 2×40 ml of pH 2 phosphate buffer, 10 ml of water, anddried over anhydrous magnesium sulfate. Evaporation of the solventleaves a yellow gum which is chromatographed on 15 g of silica gel. Thecolumn is eluted with 75 ml of benzene, 150 ml of 1:1 benzene-methylenechloride, 150 ml of methylene chloride, and 300 ml of chloroform.Evaporation of the chloroform fraction under reduced pressure affords anoil which is taken up in 10 ml of methylene chloride and stirred for 30min with a solution of 0.050 g of sodium bicarbonate in 5 ml of water.The methylene chloride phase is separated, washed with 5 ml of water,dried over anhydrous magnesium sulfate, and evaporated to givebenzhydryl 7α-ethynyl-7-(2-thienylacetamido)cephalosporanate.

D. Sodium 7α-ethynyl-7-(2-thienylacetamido)cephalosporanate

Benzhydryl 7α-ethynyl-7-(2-thienylacetamido)cephalosporanate, 0.179 g,is dissolved in 1.5 ml of anisole and treated with 4.5 ml oftrifluoroacetic acid. After having been kept at room temperature for 10min, the solution is evaporated in vacuo to remove excess reagents. Theresidue is dissolved in 10 ml of methylene chloride and is stirred for30 min with a solution of 0.045 g of sodium bicarbonate in 7.5 ml ofwater. The phases are separated; the organic layer is extracted with 4ml of water; and the combined aqueous solutions are washed with 3×4 mlof methylene chloride and lyophilized to give a powder. The crudeproduct is partially dissolved in anhydrous methanol and filtered.Evaporation of the solvent from the filtrate affords sodium7α-ethynyl-7-(2-thienylacetamido)cephalosporanate.

Following the procedures described in steps B, C, and D, benzhydryl7β-ethynyl-7-azidocephalosporanate can be converted to sodium7β-ethynyl-7-(2-thienylacetamido)cephalosporanate. The acetoxymethylgroup of this compound and of the 7β-ethynyl compound can be convertedto other 3-substituted cephalosporins as described in the foregoingexamples. The acetoxy group can be displaced by nitrogen bases and withother compounds of greater nucleophilicity than the oxygen nucleophile.Other 3-substituted compounds, such as 3-hydroxymethyl, can also beprepared and further derivitized to produce new compounds. Thecorresponding 3-desacetyl derivatives can also be prepared by utilizingbenzhydryl 7-diazodesacetoxycephalosporanate in step A of the sequencedepicted above. Similarly, other 7-acylamido compounds can be preparedusing the procedures described in other examples.

EXAMPLE 120 7α-Phenyl-7-[2-(4-pyridylthio)acetamido]cephalosporanic AcidA. Benzhydryl 7-phenyl-7-azidocephalosporanate

A stirring solution of 1.350 g of benzhydryl 7-diazocephalosporanate in15 ml each of methylene chloride and ether is cooled to -78° C. under anitrogen atmosphere and is treated dropwise over a period of 20 min witha solution of 0.727 g of triphenylboron in 20 ml of ether. The mixtureis stirred an additional 10 min at -78° C., at which time the diazocompound is completely reacted as determined by infrared analysis of asmall aliquot. Bromine azide in methylene chloride, 45 ml of a 0.28 Nsolution, is then added over a period of 30 min. The cooling bath isremoved and the mixture is stirred for a further 2 hrs. at roomtemperature.

The reaction mixture is poured into a solution of 30 ml of 0.1 Nthiosulfate and 30 ml of 0.5 M pH 7 phosphate buffer and agitated. Theorganic phase is separated, washed with water and saturated brine, driedover anhydrous magnesium sulfate, and evaporated. The crude product ischromatographed on silica gel using benzene as eluant to affordbenzhydryl 7α-phenyl-7-azidocephalosporanate and benzhydryl7β-phenyl-7-azidocephalosporanate.

B. Benzhydryl 7α-phenyl-7-aminocephalosporanate

A mixture of 0.450 g of benzhydryl 7α-phenyl-7-azidocephalosporanate and0.450 g of platinum oxide in 45 ml of dioxane is stirred under hydrogenat atmospheric pressure for one hour. After purging the system withnitrogen, an additional 0.450 g of platinum oxide is added, and thereaction mixture is again placed under hydrogen and stirred for 2 hrs.At this time the azide is completely reacted as evidenced by infraredanalysis of an aliquot. The solvent is evaporated at reduced pressureand the residue taken up in 25 ml of chloroform and filtered through apacked pad of silica gel G and diatomaceous earth in a 60 ml sinteredglass funnel. The product is eluted with chloroform until 200 ml ofsolution has been collected. Evaporation of the solvent under reducedpressure gives benzhydryl 7α-phenyl-7-aminocephalosporanate, which isacylated directly without further purification.

C. Benzhydryl 7α-phenyl-7-[2-(4-pyridylthio)acetamido]cephalosporanate

To a stirring solution of 0.395 g of benzhydryl7α-phenyl-7-aminocephalosporanate in 15 ml of dry methylene chloride isadded 0.130 g of (4-pyridylthio)acetic acid, followed by 5 ml ofdimethylforamide to effect solution. Dicyclohexylcarbodiimide, 0.159 g,is then added, and the mixture is stirred at room temperature and undera nitrogen atmosphere for 18 hrs. The precipitate of dicyclohexylurea isremoved by filtration and the filtrate evaporated under high vacuum. Theresidue is dissolved in 25 ml of chloroform and washed with water, 2%sodium bicarbonate, water, and saturated brine. The chloroform is driedover anhydrous magnesium sulfate and evaporated under reduced pressureto give the crude product. This material is chromatographed on 20 g ofsilica gel using an ethyl acetate in benzene gradient as eluant toafford benzhydryl7α-phenyl-7-[2(4-pyridylthio)acetamido]cephalosporanate.

D. 7α-Phenyl-7-[2(4-pyridylthio)acetamido]cephalosporanic-acid

To a stirring solution of 0.330 g of benzhydryl7α-phenyl-7-[2(4-pyridylthio)acetamido]cephalosporanate in 2.5 ml ofanisole is added 7 ml of trifluoroacetic acid. The reaction mixture isstirred for 10 min at room temperature. Excess anisole andtrifluoroacetic acid are removed under high vacuum, and the residue istaken up in 20 ml of chloroform and layered with 20 ml of watercontaining 0.100 g of sodium bicarbonate. The mixture is stirred for 30min at room temperature, and the organic phase is separated and washedwith water. The combined aqueous phase is washed with methylenechloride, layered with ethyl acetate, and acidified to pH 3 with 2 Nhydrochloric acid. The ethyl acetate phase is separated, dried overanhydrous sodium sulfate, and evaporated to afford7α-phenyl-7-[2-(4-pyridylthio)acetamido]cephalosporanic acid.

Following the procedures described in steps B-D, benzhydryl7β-phenyl-7-azidocephalosporanate is converted to7β-phenyl-7-[2-(4-pyridylthio)acetamido]cephalosporanic acid.

The acetoxymethyl group of both 7β- and7β-phenyl-7-[2(4-pyridylthio)acetamido]cephalosporanate can be convertedto other 3-substituents using the procedures of the foregoing examples.The acetoxy group can be displaced by nitrogen bases and other compoundsof greater nucleophilicity than the oxygen nucleophile. The3-acetoxymethyl can be catalytically reduced in the presence of hydrogento produce the 3-methyl derivatives. Other 3-substituted compounds, suchas 3-hydroxymethyl, can also be made and converted to other usefulcompounds. Also, the benzhydryl 7α-phenyl-7-aminocephalosporanate can beacylated with other acylating agents such as phenyl acetyl chloride,thienylacetylchloride, furylacetylchloride, and the like to produce thecorresponding 7-acylamido compounds using the procedures described inthe other examples hereof.

EXAMPLE 121 7α-Ethyl-7-(D-2-amino-α-phenylacetamido)cephalosporanic acidA. Benzhydryl 7-ethyl-7-azidocephalosporanate

A solution of 1.350 g of benzhydryl 7-diazocephalosporanate in 15 mleach of methylene chloride and ether is cooled to -78° C. under anitrogen atmosphere and stirred while 0.295 g of triethylboron in 15 mlof ether is added dropwise over a period of 15 min. The mixture isstirred an additional 5 min. at -78° C., at which time the diazocompound is completely reacted as determined by infrared analysis of asmall aliquot. Bromine azide in methylene chloride, 45 ml of a 0.28 Nsolution, is then added dropwise over a period of 30 min. The coolingbath is removed and the mixture is stirred for an additional 2 hours atroom temperature. The reaction mixture is poured into a solution of 30ml each of 0.1 N sodium thiosulfate and 0.5 M pH 7 phosphate buffer andagitated. The organic phase is separated, washed with water andsaturated brine, dried over anhydrous magnesium sulfate, and evaporated.The residue is chromatographed on silica gel using benzene as eluant togive benzhydryl 7α-ethyl-7-azido-cephalosporanate and benzhydryl7β-ethyl-7-azidocephalosporanate.

B. Benzhydryl 7α-ethyl-7-aminocephalosporanate

A solution of 0.520 g of benzhydryl 7α-ethyl-7-azidocephalosporanate in50 ml of dioxane is treated with 0.520 g of platinum oxide, and themixture is stirred under hydrogen at atmospheric pressure for one hour.After purging the mixture with nitrogen, an additional 0.520 g ofplatinum oxide is added. The mixture is again placed under hydrogen andstirred for 2 hours, at which time the azide is completely reacted asdetermined by infrared analysis of an aliquot. The dioxane is removedunder reduced pressure and the residue taken up in 30 ml of chloroformand filtered through a packed pad of silica gel G and diatomaceous earthin a 60 ml sintered glass funnel. The product is eluted with chloroformuntil 200 ml of solution has been collected. Evaporation of the solventunder reduced pressure gives benzhydryl7α-ethyl-7-aminocephalosporanate.

C. Benzhydryl 7α-ethyl-7-(D-2-azido-2-phenylacetamido) cephalosporanate

To a stirring solution of 0.418 g of benzhydryl7α-ethyl-7-aminocephalosporanate in 20 ml of dry methylene chloride at0° C. is added rapidly 0.350 g of D-α-azidophenylacetic acid, followedby 0.30 ml of pyridine 60 seconds later. The reaction mixture is stirredfor 30 min. at 0° C. and then poured onto 40 ml of water. The mixture isagitated, and the methylene chloride phase is separated and washedsuccessively with water, dilute sodium bicarbonate, water, and saturatedbrine. After having been dried over anhydrous magnesium sulfate, themethylene chloride is removed under reduced pressure to yield the crudeproduct. This material is chromatographed on 40 g of silica gel using achloroform in benzene gradient as eluant to afford benzhydryl7α-ethyl-7-(D-2-azido-2-phenylacetamido)cephalosporanate.

D. 7α-Ethyl-7-(D-2-azido-2-phenylacetamido)cephalosporanic acid

A solution of 0.388 g of benzhydryl7α-ethyl-7-(D-2-azido-2-phenylacetamido)cephalosporanate in 1.0 ml ofanisole is cooled in an ice bath and treated with 4.0 ml oftrifluoroacetic acid. The reaction mixture is kept at 0° for 10 min.with occasional swirling, and then evaporated in vacuo to remove excessanisole and trifluoroacetic acid. The residue is taken up in 20 ml ofmethylene chloride and extracted with three 5 ml portions of saturatedsodium bicarbonate solution. The combined aqueous extracts are washedwith two 5 ml portions of methylene chloride, acidified to pH 2 with 2.5N hydrochloric acid, and extracted with four 10 ml portions of ethylacetate. The organic extracts are combined and dried over anhydrousmagnesium sulfate. Evaporation of the solvent under reduced pressureleaves 7α-ethyl-7-(D-2-azido-2-phenylacetamido)cephalosporanic acid.

E. 7α-Ethyl-7-(D-2-amino-2-phenylacetamido)cephalosporanic acid

0.291 g of 7α-ethyl-7-(D-2-azido-2-phenylacetamido)cephalosporanic acidis taken up in 3.0 ml of acetic acid and 4.5 ml of water, cooled in anice bath, stirred, and treated with 1.550 g of zinc dust. The mixture isstirred for 10 min. and then filtered through sintered glass. The zincis washed with 20 ml of water. The filtrate and washings are combined,saturated with hydrogen sulfide, and filtered through a pad ofdiatomaceous earth. Lyophilization of the filtrate affords crude7α-ethyl-7-(D-2-amino-2-phenylacetamido)cephalosporanic acid. Thismaterial is freed of acetic acid by repetitive dissolution in water andlyophilization.

In an analogous manner, 7β-ethyl-7-azidocephalosporanate can beconverted to 7β-ethyl-7-(D-2-amino-2-phenylacetamido)cephalosporanicacid by procedures C-E. Substitution of L-α-azidophenylacetic acid andDL-α-azidophenylacetic acid in Step C and subsequent transformationsgives 7α-ethyl-7-(L-2-amino-2-phenylacetamido) cephalosporanic acid,7β-ethyl-7-(L-2-amino-2-phenylacetamido)cephalosporanic acid,7α-ethyl-7-(DL-2-amino-2-phenylacetamido)-cephalosphoranic acid, and7β-ethyl-7-(DL-2-amino-2-phenylacetamido)-cephalosphoranic acid.Similarly using other acylating agents in C above affords analogous7-acylamido compounds.

The acetoxymethyl group of these compounds can be converted to other3-substituents in the usual manner. The acetoxy group can be displacedby nitrogen bases and other compounds of greater nucleophilicity thanthe oxygen nucleophile. Other 3-substituted compounds, such as3-hydroxymethyl, can also be prepared and converted to otherderivatives. In addition, the 3-acetoxymethyl group can be catalyticallyreduced in the presence of hydrogen to afford the 3-methylcephalosporins.

EXAMPLE 122 A. Benzhydryl 7-benzyl-7-azidocephalosporanate

To a solution of 0.900 g (0.002 m) of benzhydryl 7-diazocephalosporanatein 10 ml of CH₂ Cl₂ and 10 ml of ether, cooled to -78° C. under a N₂atmosphere is added dropwise over 15 minutes, 10 ml of a 0.02 Mtribenzyl boron solution in ether.

A check on a small aliquot shows the absence of the diazo compound byir. The reaction mixture is stirred a further 5 minutes at -78° C. andis then treated with 40 ml of a 0.3 N solution of BrN₃ in methylenechloride. The reaction mixture is stirred at -78° C. for 15 minutes andthen allowed to come to room temperature over 45 minutes and stirredanother 20 minutes at room temperature. The reaction mixture is pouredinto ice cold 5% NaHCO₃ solution, agitated and the organic phase isseparated and washed once with N/10 thiosulfate (30 ml) and then withwater, dried and evaporated. The residue is chromatographed on 30 g ofsilica gel to give benzhydryl 7α-benzyl-7-azidocephalosporanate andbenzhydryl 7β-benzyl-7-azidocephalosporanate.

B. Benzhydryl 7α-benzyl-7-aminocephalosporanate

0.500 g of benzhydryl 7-azido-7α-benzylcephalosporanate is dissolved in25 ml of ethyl acetate and 0.500 g of PtO₂ is added and the mixture isstirred overnight under H₂. The catalyst is filtered off, the filtrateis evaporated and the residue is chromatographed on silica gel (15 g) togive the product.

C. Benzhydryl 7α-benzyl-7-(p-carboxymethylphenylacetamido)cephalosporanate

0.400 g of benzhydryl 7α-benzyl-7-aminocephalosporanate is dissolved in25 ml of CH₂ Cl₂ and cooled to 0° C. 0.400 g of p-phenylenediacetylchloride is added followed 60 seconds layer by 0.400 g of pyridine. Thereaction mixture is agitated, the organic phase is separated and washedonce with pH 2 phosphate buffer and then with water. The organic phaseis dried and evaporated. The crude product so obtained is purified bychromatography over silica gel.

D. Disodium 7α-benzyl-7-(p-carboxymethylphenylacetamido)cephalosporanate

0.325 g of benzhydryl7α-benzyl-7-(p-carboxymethylphenylacetamido)-cephalosporanate isdissolved in 3 ml of anisole and treated with 10 ml of trifluoroaceticacid at room temperature for 10 minutes. The trifluoroacetic acid andthe anisole are evaporated under reduced pressure below 40°. The residueis taken up in 25 ml of ethyl acetate (freed of any acetic acid) andtreated with 20 ml of H₂ O containing 0.120 g of NaHCO₃. The mixture isstirred for 1/2 hour and the organic phase is separated and washed withwater. The combined aqueous phase is washed twice with ethyl acetate andthen freeze dried to give the product which is further purified byrecrystallization from MeOH/iPrOH.

Reaction of benzhydryl 7β-benzyl-7-azidocephalosporanate in the abovesequence gives7β-benzyl-7-(p-carboxymethylphenylacetamido)cephalosporanate.

When other acylating agents such as phenylacetyl chloride,2-thienylacetyl chloride, 2-furylacetyl chloride are used in Step Chereof the corresponding 7-acylamido compounds are obtained inaccordance with the procedures of the examples hereof. Thecephalosporins obtained are converted to other 3-substituted productssuch as the 3-methyl, 3-hydroxy methyl, and 3-pyridinummethyl compoundsin accordance with methods well known in this art and described in theforegoing examples.

EXAMPLE 123 7α-Vinyl-7-(phenylacetamido)cephalosporanic acid A.Benzhydryl-7-vinyl-7-azidocephalosporanate

0.900 g (0.002 m) of benzhydryl-7-diazocephalosporanate is dissolved in10 ml CH₂ Cl₂ and 10 ml Et₂ O, cooled to -78° C., under N₂ and treateddropwise over 15 minutes with 10 ml of a 0.02 M trivinyl boron solutionin ether. A check on a small aliquot shows the absence of the diazocompound by ir. The reaction mixture is stirred at -78° C. for a further5 minutes and then treated with 40 ml of a 0.3 N solution of BrN₃ in CH₂Cl₂. The reaction mixture is stirred at -78° C. for 15 minutes andallowed to come to room temperature over 45 minutes and then stirredanother 20 minutes at room temperature.

The reaction mixture is poured into ice cold 5% NaHCO₃ solution,agitated and the organic phase is separated, washed once with 0.1 Nsodium thiosulfate solution (30 ml) and then with water, dried andevaporated. The residue is chromatographed on 30 g of silica gel to givebenzhydryl 7α-vinyl-7-azidocephalosporanate and benzhydryl7α-vinyl-7-azidocephalosporanate.

B. Benzyhdryl 7α-vinyl-7-aminocephalosporanate

0.030 g of anhydrous cobaltous bromide is dissolved in 50 ml of absoluteethanol and treated with 0.065 g of α,α'-dipyridyl. 0.300 g of thebenzhydryl 7α-vinyl-7-azidocephalosporanate is then added dissolved in 2ml of EtOH, followed by 0.080 g of sodium borohydride. After the rapidevolution of gas ceases (˜15 minutes) the reaction mixture is pouredinto pH 7 phosphate buffer and extracted with methylene chloride, washedonce with water, dried and evaporated. The residue is chromatographed onsilica gel (10 g) to give benzhydryl 7α-vinyl-7-aminocephalosporanate.

C. Benzhydryl 7α-vinyl-7-(phenylacetamido)cephalosporanate

0.450 g of benzhydryl 7α-vinyl-7-aminocephalosporanate is dissolved in25 ml of methylene chloride and cooled to 0°. 0.400 ml of phenylacetylchloride is added followed by 0.400 ml of pyridine. The reaction mixtureis stirred for 25 minutes at 0° C. and poured onto crushed ice. Themixture is agitated, the organic phase is separated and washed with pH 2phosphate buffer and then with water. The organic phase is dried andevaporated. The residue is chromatographed on silica gel to give thepurified product.

D. 7α-Vinyl-7-(phenylacetamido)cephalosphoranic acid

0.350 g of benzhydryl 7α-vinyl-7-(phenylacetamido)cephalosporanate isdissolved in 3 ml of anisole and treated with 10 ml of trifluoroaceticacid at room temperature for 10 minutes. The trifluoroacetic acid andanisole are evaporated under reduced pressure below 40° C. The residueis taken up in methylene chloride (25 ml) and stirred for 1/2 hour with20 ml of water containing 0.120 g of NaHCO₃. The organic phase isseparated and washed once with water. The combined aqueous phase istaken to pH 2 and extracted with ethyl acetate. The ethyl acetateextract is dried and evaporated to give the crude product which ispurified by crystallization from ethyl acetate/ether.

Reaction of benzhydryl 7β-vinyl-7-azidocephalosporanate in the abovesequence gives 7β-vinyl-7-(phenylacetamido)cephalosporanic acid.

EXAMPLE 124 A. Benzhydryl7α-aminomethyl-7-(2-benzhydryloxycarbonylphenylacetamido)-cephalosporanate

0.800 g of benzhydryl7α-cyano-7-(2-benzhydryloxycarbonylphenylacetamido)cephalosporanate(prepared as described in Example 108C) is dissolved in 10 ml oftetrahydrofuran and cooled to 0° C. 1 ml of a 1 M solution of BH₃.THF inTHF is added dropwise over 5 minutes and the mixture is stirred at 0°for 1/2 hour.

The reaction mixture is poured into pH 7 phosphate buffer and extractedwith methylene chloride. The methylene chloride extract is dried andevaporated to give crude product which is purified by chromatography onsilica gel.

B. Disodium 7α-aminomethyl-7-(2-carboxyphenylacetamido)cephalosporanate

0.450 g of benzhydryl7α-(aminomethyl)-7-(2-benzhydryloxycarbonylphenylacetamido)cephalosporanateis dissolved in 3 ml of anisole, 10 ml of CF₃ COOH is added and thereaction mixture is allowed to stand at room temperature for 10 minutes.The anisole and CF₃ COOH are removed under reduced pressure below 40° C.The residue is taken up in 25 ml ethyl acetate and treated with 15 ml ofH₂ O containing 0.200 g of NaHCO₃. The mixture is stirred 15 minutes.The organic phase is evaporated and washed once with water. The combinedaqueous phase is washed once with methylene chloride and then freezedried. The product so obtained is purified by crystallization frommethanol-isopropanol mixtures.

Reaction of the benzhydryl7β-cyano-7-(2-benzhydryloxycarbonylphenylacetamido)cephalosporanate inthe above sequence gives the7β-aminomethyl-7-(2-carbonylphenylacetamido)cephalosporanate.

EXAMPLE 125 Sodium7-hydroxymethyl-7-(2-thienylacetamido)cephalosporanate A. Benzhydryl7-(4-nitrobenzilidinamino)cephalosporanate

An equimolar mixture of benzhydryl 7-aminocephalosporanate and4-nitrobenzaldehyde is heated under nitrogen in 200 ml of benzene pergram aldehyde, and the water azeotropically removed over a one hourperiod. The solution is evaporated under reduced pressure to give afoam. The ir (CHCl₃) shows bands at 5.60 (β-lactam) and 5.75μ (esters),while the NMR (60 Hz) shows peaks at (numbers are in Hz from internalTMS in CDCl₃) 518,516 (1H), 596, 587, 575, 566 (AB quartet; 4H), 439(10H), 416 (1H), 330, 328, 325, 323 (doublet of doublets; 1H), 311, 306(1H), 308, 295, 288, 274 (AB quartet; 2H), 227, 209, 206, 187 (ABquartet; 2H), and 119 (3H). Thin layer chromatography on 250μ silicaplates with 10% ethyl acetate in chloroform shows essentially one spotat Rf≅0.58; only traces of the starting materials can be detected.

B. Benzhydryl 7-hydroxymethyl-7-(4-nitrobenzilidinamino)cephalosporanate

A gentle stream of nitrogen is passed into a half-dram vial containing60 mg of benzhydryl 7-(4-nitrobenzilidinamino)cephalosporanate and aftera few minutes 0.3 ml of N,N-dimethylformamide is added. The nitrogenstream is continued, bubbling through the greenish brown solution for ca30 seconds, and then a stream of formaldehyde gas in nitrogen, generatedby heating ˜15 mg of paraformaldehyde in a nitrogen stream, is passedthrough. The color is discharged and the resultant solution isevaporated to a gum under high vacuum. The gum is flushed by dissolvingit in a small volume of chloroform and again evaporating to a gum underhigh vacuum. The product exhibits an ir (neat) spectrum with hydroxy,β-lactam, and ester absorption. The nmr spectrum in CDCl₃ shows theexpected singlet for the benzilidine proton, and new absorptionassociated with the hydroxymethyl group.

C. Benzhydryl 7-hydroxymethyl-7-aminocephalosporanate tosylate salt

A mixture of 100 mg of powdered 2,4-dinitrophenylhydrazine, 85.5 mg ofp-toluene sulfonic acid monohydrate, and 3 ml of absolute ethanol arestirred for 30 minutes. To this is added a solution of 304 mg ofbenzhydryl 7-hydroxymethyl-7-(4-nitrobenzilidinamino) cephalosporanatein 3 ml of ethanol and 0.5 ml of methylene chloride. The mixture isstirred for 30 minutes, filtered, and after the filter cake has beenthoroughly washed with ethanol, the filtrates are evaporated underreduced pressure at or below ambient temperature. The resultant solid iswashed several times with ether and dried in a nitrogen stream. The irand nmr of the product are consistent with the assigned structure.

D. Benzhydryl 7-hydroxymethyl-7-aminocephalosporanate

A mixture of 3.5 ml of ether, 0.5 ml of ethyl acetate, 2 ml of water and22 mg of dipotassium hydrogen phosphate is prepared. To this is added100 mg of benzhydryl 7-hydroxymethyl-7-aminocephalosporanate tosylatesalt and the mixture is shaken vigorously for several minutes. Afterphase separation the aqueous phase is again extracted with ether, thecombined organic phases are dried with anhydrous magnesium sulfate, andevaporated to a gum under reduced pressure. The product is flushedseveral times by dissolving it in a small volume of chloroform and againevaporating to a gum under high vacuum. The product so obtained exhibitsir and nmr spectra consistent with the assigned structure.

E. Benzhydryl7-(2-thienylacetoxymethyl)-7-(4-nitrobenzilidinamino)cephalosporanate

A solution of 90 mg of benzhydryl7-hydroxymethyl-7-(4-nitrobenzilidinamino)-cephalosporanate in 0.3 ml ofdry methylene chloride is cooled to 0° and treated with 0.5 ml of drymethylene chloride containing 100 mg of pyridine, also cooled to 0°. Tothis is added with cooling and stirring over ten minutes a cooledsolution of 25 mg of 2-thienylacetyl chloride in 0.25 ml of drymethylene chloride, and held for 2 hours at 0°. The mixture is thenshaken with a solution of 55 mg of dipotassium hydrogen phosphate in 3ml of water, the organic phase is removed, dried with anhydrousmagnesium sulfate and taken to a gum under high vacuum. The gum isflushed by dissolving it in a small volume of chloroform and againevaporating it to a gum under reduced pressure. The product is purifiedby preparative tlc on 1000μ silica plates with fluoroscent indicator.After development with 5% ethyl acetate in chloroform, the desired bandis located with the aid of both short and long wave uv light, removed,and eluted with ethyl acetate. The product exhibits ir and nmr spectraconsistent with the proposed structure.

F. Benzhydryl 7-(2-thienylacetoxymethyl)-7-aminocephalosporanate

A mixture of 15.9 mg of powdered 2,4-dinitrophenylhydrazine, 13.5 mg ofp-toluene sulfonic acid, and 2 ml of absolute ethanol is stirred for 30minutes. To this is added a solution of 58 mg of benzhydryl7-(2-thienylacetoxymethyl)-7-(4-nitrobenzilidinamino)cephalosporanate in1.5 ml of ethanol and 0.2 ml of methylene chloride. After stirring for30 minutes the mixture is filtered, and the cake washed thoroughly withethanol. The filtrate is evaporated under reduced pressure at or belowambient temperature and the resultant solids washed several times withether. The solids are shaken with a mixture of 28 mg of dipotassiumhydrogen phosphate, 2 ml of water and 4 ml of ether, the phases areseparated, and the aqueous phase is again extracted with ether. Thecombined organic phases are dried with anhydrous magnesium sulfate, andevaporated to a gum under high vacuum. The product is purified bypreparative tlc on 1000μ silica plates with fluorescent indicator using30% ethyl acetate in benzene as eluent. After locating the desired bandwith the help of both short and long wave length uv light, it is removedand eluted with ethyl acetate. The product exhibits the desired ir andnmr absorptions, and is essentially homogeneous by tlc.

G. Benzhydryl 7-hydroxymethyl-7-(2-thienylacetamido)cephalosporanateI--By O→N acyl migration

Benzhydryl 7-(2-thienylacetoxymethyl)-7-aminocephalosporanate undergoesspontaneous O to N acyl migration. When the migration has proceeded to asatisfactory extent as judged by tlc, the substantially more polarproduct, benzhydryl7-hydroxymethyl-7-(2-thienylacetamido)cephalosporanate, may be isolatedby preparative tlc or chromatography on silica. It exhibits all of thedesired characteristics in the ir and nmr spectra and is essentiallyhomogeneous by tlc.

II--By Direct Acylation with Anhydride

A mixture of 100 mg of benzhydryl7-hydroxy-methyl-7-aminocephalosporanate tosylate salt and 43 mg of2-thienylacetic anhydride is shaken vigorously with a mixture of 2 ml ofwater, 3.5 ml of ether, 0.5 ml of ethyl acetate and 30 mg of dipotassiumhydrogen phosphate. The phases are separated and the aqueous phaseextracted again with ether, the organic phases are combined, dried withmagnesium sulfate, and concentrated to ca 0.5 ml. After adding 0.1 ml ofpyridine the reation mixture is allowed to stand for 18 hours at roomtemperature and evaporated to an oil under high vacuum. The oil is takenup in 5 ml of ether and shaken with a mixture of 25 mg of dipotassiumhydrogen phosphate in 2 ml of water. After phase separation andreextraction of the aqueous phase with 3 ml of ether, the combinedorganic phases are dried with anhydrous magnesium sulfate and evaporatedto an oil under high vacuum. The crude product is flushed twice bydissolving it in a small volume of chloroform and again evaporatingunder high vacuum, after which it is purified by preparative tlc. Thematerial so obtained is identical in all respects to that obtained bythe rearrangement procedure.

III--By Direct Acylation with Acid Chloride

A solution of benzyl 7-hydroxymethyl-7-aminocephalosporanate (preparedfrom 114 mg of tosylate salt) in 0.2 ml of sieve dried methylenechloride is cooled to 0°. To this is added dropwise with stirring, 33 mgof 2-thienylacetyl chloride in 0.2 ml of sieve dried methylene chlorideover 30 seconds, followed by dropwise addition of 16 mg of pyridine alsoin 0.2 ml of sieve dried methylene chloride. The mixture is stirred for1 hour at 0°, then evaporated to a gum under a stream of dry nitrogen.The gum is shaken vigorously with a mixture of 77 mg of dipotassiumhydrogen phosphate, 3.5 of ether, 0.5 ml of ethyl acetate, and 2 ml ofwater. After phase separation the aqueous layer is again extracted withether and the combined organic phases dried with anhydrous magnesiumsulfate, filtered and evaporated under reduced pressure. The resultantgum is purified by preparative tlc on 1000μ silica plates withfluorescent indicator. The desired band is located by means of shortwave length uv light, removed and eluted with ethyl acetate. The productexhibits all the physical and spectral properties expected for thedesired compound.

H. Sodium 7-hydroxymethyl-7-(2-thienylacetamido)cephalosporanate

A mixture of 59 mg of benzhydryl7-hydroxymethyl-7-(2-thienylacetamido)cephalosporanate, 0.5 ml ofanisole, and 1.0 ml of trifluoroacetic acid is allowed to stand at roomtemperature for 10 minutes, after which the mixture is concentratedunder reduced pressure to an oil. The product is dissolved in 5 ml ofchloroform and shaken with a mixture of 5 ml of water and 8.4 mg ofsodium bicarbonate. The phases are separated and the organic phase againwashed with water. The combined aqueous phases are washed with methylenechloride and then lyophyllyzed to give 42 mg of solid, exhibiting ir andnmr spectral features consistent with the desired product.

EXAMPLE 126 Sodium 7-methoxymethyl-7-(2-furylacetamido)-cephalosporanateA. Benzhydryl 7-hydroxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate

To an ice cold, stirring solution of 0.350 g of benzhydryl7-hydroxymethyl-7-aminocephalosporanate in 1.0 ml of dry methylenechloride is added dropwise over a period of 30 seconds 0.119 g of(2-furyl)acetyl chloride in 1.0 ml of dry methylene chloride, followedby 0.065 g of pyridine in 0.5 ml of dry methylene chloride. The reactionmixture is stirred for 60 minutes at 0°, then poured into 10 ml ofmethylene chloride layered with 10 ml of ice water and agitated. Theorganic phase is separated, washed with dilute aqueous dipotassiumhydrogen phosphate solution, water, and saturated brine, and dried overanhydrous magnesium sulfate. Evaporation of the methylene chloride underreduced pressure yields a gum which is purified by preparative tlc on1000μ silica gel GF plates. The desired band is visualized by shortwavelength uv light, removed, and eluted with ethyl acetate. Evaporationof the ethyl acetate under reduced pressure gives benzhydryl7-hydroxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate.

B. Benzhydryl 7-methoxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate

A stirring solution of 0.334 g of benzhydryl7-hydroxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate in 2 ml ofmethylene chloride is kept at -5° while 7.5μ of boron trifluorideetherate is added. A solution of diazomethane in methylene chloride isthen added slowly at the same temperature. A vigorous reaction occurswith the formation of a white solid (polymethylene), and the addition isstopped when a faint yellow color persists in the solution for a shorttime. After 30 minutes at -5°, the solid is filtered off and thefiltrate is diluted with 10 ml of methylene chloride and washed withdilute sodium bicarbonate and water, and dried over anhydrous magnesiumsulfate. Evaporation of the solvent under reduced pressure yields an oilwhich is chromatographed on silica gel using chloroform as eluant toafford benzhydryl7-methoxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate.

C. Sodium 7-methoxymethyl-7-(2-furylacetamido)cephalosporanate

A solution of 0.295 g of benzhydryl7-methoxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate in 2.5 ml ofanisole is treated with 7.5 ml of trifluoroacetic acid and then kept atroom temperature for 10 minutes. Excess reagents are removed underreduced pressure at ambient temperature. The residue is dissolved in 20ml of methylene chloride and stirred with a solution of 0.075 g ofsodium bicarbonate in 10 ml of water. After phase separation, themethylene chloride layer is extracted with 5 ml of water. The combinedaqueous solutions are washed with three 5 ml portions of methylenechloride and lyophilized to give sodium7-methoxymethyl-7-[2-(2-furyl)acetamido]cephalosporanate as a powder.

The acetoxymethyl group of sodium7-methoxymethyl-7-[2-(2-furyl)-acetamido]cephalosporanate can beconverted to other 3-substituted cephalosporins. The acetoxy group canbe displaced by reaction with nitrogen bases and other compounds ofgreater nucleophility than the oxygen nucleophile. Further, the3-acetoxymethyl can be catalytically reduced in the presence of hydrogento produce the 3-methyl cephalosporin. Other 3-substituted compounds,such as 3-hydroxymethyl, can also be made and converted to othervaluable compounds.

EXAMPLE 127 Disodium7-carboxymethyl-7-(2-thienylacetamido)cephalosporanate A.2,2,2-Trichloroethyl-7-(4-nitrobenzaldimino)cephalosporanate

0.800 g of 2,2,2-trichloroethyl-7-aminocephalosporanate is dissolved in200 ml C₆ H₆, 0.300 g of p-nitrobenzaldehyde is added. The mixture isrefluxed with azeotropic removal of the water formed. After 1 hour thebenzene solution is evaporated under reduced pressure to give a foam.

B.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-(4-nitrobenzaldimino)cephalosporanate

0.550 g of 2,2,2-trichloroethyl-7-(4-nitrobenzaldimino)cephalosporanateis dissolved in 5 ml of DMF under N₂. 0.205 g of2,2,2-trichloroethylglyoxalate in 2 ml of DMF is added dropwise over 15minutes. The reaction mixture is stirred for 1/2 hour and the DMF isremoved under reduced pressure to give the product.

C.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-aminocephalosporanate

A mixture of 0.200 g of 2,4-dinitrophenylhydrazine, 0.171 g of p-toluenesulfonic acid monohydrate and 6 ml of absolute ethanol is stirred for 30minutes. To this is added a solution of 0.600 g of2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-(4-nitrobenzaldimino)cephalosporanatein 6 ml of ethanol and 0.5 ml of CH₂ Cl₂. The mixture is stirred for 30minutes, filtered and after the filter cake has been thoroughly washedwith ethanol, the filtrate and washings are evaporated under reducedpressure to give a solid.

The solid is suspended in 15 ml of water containing 155 mg of K₂ HPO₄and extracted thrice with methylene chloride (20 ml). The combinedorganic phase is dried over MgSO₄ and evaporated to a gum under reducedpressure to give the product.

D.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-(2-thienylacetamido)cephalosporanate

0.620 g of2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-aminocephalosporanateis dissolved in 100 ml of CH₂ Cl₂, cooled to 0° and treated with 0.160 gof 2-thienylacetyl chloride, followed by dropwise addition of 0.100 gpyridine in 2 ml of CH₂ Cl₂. The mixture is stirred for 1 hour at 0° andpoured onto crushed ice. After agitation the organic phase is separatedand washed once with pH 2 phosphate buffer and once with water. Afterdrying over MgSO₄ the solvent is removed to give the crude product,which is purified by chromatography over silica gel.

E.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonylmethylsulfonyloxymethyl)-7-(2-thienylacetamido)cephalosporanate

0.350 g of2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonylhydroxymethyl)-7-(2-thienylacetamido)cephalosporanateis dissolved in 10 ml of CH₂ Cl₂ and and cooled to 0°, 0.115 g methanesulfonylchloride is added and then 0.080 g of pyridine. The reactionmixture is stirred at 0° for 11/2 hours. The reaction mixture is pouredonto crushed ice and agitated. The organic phase is separated and washedwith pH 2 phosphate buffer, and water and is dried and evaporated. Thecrude product is purified by chromatography on silica gel.

F.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonyliodomethyl)-7-(2-thienylacetamidocephalosporanate)

0.300 g of2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonylmethylsulfonyloxymethyl)-7-(2-thienylacetamido)cephalosporanateis dissolved in 10 ml of DMSO. Sodium iodide 0.100 g is added and themixture stirred at room temperature overnight. The dimethyl sulfoxide isremoved under reduced pressure. The residue is partitioned between 10 mlof H₂ O and 10 ml CH₂ Cl₂. The organic phase is washed once with waterdried and evaporated to give the crude product which is purified bypreparative tlc on silica gel plates.

G.2,2,2-Trichloroethyl-7-(2,2,2-trichloroethoxycarbonylmethyl)-7-(2-thiophenacetamido)cephalosporanate

0.200 g2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonyliodomethyl)-7-(2-thiophenacetamido)cephalosporanateis dissolved in 20 ml ethanol, 0.100 g of sodium acetate and 0.100 g of5% Pd on calcium carbonate are added and the mixture is treated with H₂at 40 psig for 2 hours. The catalyst is filtered off. The filtrate isevaporated under reduced pressure and the residue is chromatographed onsilica gel to give the product.

H. Disodium 7-carbomethoxy-7-(2-thienylacetamido)cephalosporanate

0.300 g of2,2,2-trichloroethyl-7-(2,2,2-trichloroethoxycarbonylmethyl)-7-(2-thiophenacetamido)cephalosporanateis dissolved in 15 ml of 95% acetic acid. 0.300 g of Zn dust is added in5 equal amounts over 20 minutes while the mixture is vigorously stirredat room temperature. The reaction mixture is carefully poured into icecold pH 2 phosphate buffer and extracted with ethyl acetate. The ethylacetate extract is washed once with water dried and evaporated. Theresidue is taken up in methyl isobutyl ketone 10 ml and extracted with15 ml of H₂ O containing 0.120 g of NaHCO₃. The organic phase isseparated and washed once with water. The combined aqueous phase iswashed twice with methylene chloride and then freeze dried to give thecrude product. Crystallization from MeOH/iPrOH gives the pure product.

EXAMPLE 128 A. Benzhydryl 7-azido-7-trifluoromethylcephalosporanate

A degassed solution of benzhydryl 7-diazocephalosporanate, 45 mg, and144 mg CF₃ I in 15 ml benzene saturated with triethylammonium azide isirradiated with uv light >300 nm is a pyrex flask until the diazo bandat 4.8μ in the ir spectrum is consumed. The resulting solution is washedwith water, dried with MgSO₄, evaporated and carefully chromatographedon silica gel, eluting with chloroform, to afford both epimers ofbenzhydryl 7-azido-7-trifluoromethylcephalosporanate.

B. Benzhydryl 7β-amino-7α-trifluoromethylcephalosporanate Benzhydryl7β-(2-thienylacetamido)-7α-trifluoromethylcephalosporanate

Benzhydryl 7β-azido-7α-trifluoromethylcephalosporanate, 542 mg, ishydrogenated at 40 psi for three hours using 1 g PtO₂ in 50 ml dioxanecontaining 500 mg thienylacetic anhydride. The 7β-amino compound isformed during the hydrogenation and acylated in situ. After filtrationof the catalyst, the solution is treated for 15 minutes with 0.5 mlwater to hydrolyze excess anhyride, then vacuum stripped. The crudeproduct is taken up in 25 ml ethyl acetate, washed with aqueousbicarbonate, dried with MgSO₄, filtered, evaporated, and chromatographedon silica gel, using 4:1 chloroform:ethyl acetate, to afford benzhydryl7β-(2-thienylacetamido)-7α-trifluoromethylcephalosporanate. The otherepimer affords the epimeric compound by the same procedure.

C. Sodium 7β-(2-thienylacetamido)-7α-trifluoromethylcephalosporanate

Benzhydryl 7β-(2-thienylacetamido)-7α-trifluoromethylcephalosporanate,640 mg, is dissolved in 0.8 g anisole and cooled to 0° C.Trifluoroacetic acid, 4 ml, precooled to 0° C., is added and thereaction allowed to proceed for two minutes at 0° C. Vacuum of 0.1 mm isimmediately applied and the reaction mixture allowed to warm to roomtemperature without external heating. Anisole is then distilled out at30° C./0.1 mm. A few ml anisole is added to the residue and pulled offat 30° C./0.1 mm to insure total removal of trifluoroacetic acid. Theproduct is treated with 5 ml water containing 84 mg NaHCO₃ andlyophilized. The resulting powder is washed thoroughly with ether anddried, affording the desired sodium salt. The other epimer affords theepimeric sodium salt by the same procedure.

EXAMPLE 129 Sodium7α-difluoromethyl-7-(2-thienylacetamido)cephalosporanate A. Benzhydryl7β-(p-nitrobenzylideneamino)-7α-difluoromethylcephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate 571 mg, isdissolved in 20 ml of 1,2-dimethoxyethane (DME) containing 1 ml ofchlorodifluoromethane. With vigorous stirring under nitrogen, 112 mgpotassium t-butoxide is slowly introduced as a slurry in DME over aboutone hour.

After stirring an additional hour, KCl is filtered off and the volatilesremoved in vacuo to afford benzhydryl7β-(p-nitrobenzylideneamino)-7α-difluoromethylcephalosporanate. It canbe purified, if desired, by chromatography on silica gel, eluting with2% ethyl acetate in chloroform.

B. Sodium 7α-difluoromethyl-7-(2-thienylacetamido)cephalosporanate

Removal of the Schiff base functionality with aniline hydrochloride,acylation of the resulting 7-aminocephalosporin with thienylacetylchloride (or other acid chloride or anhydride), and deblocking of the4-carboxy group with trifluoroacetic acid are all accomplished in thesame manner as that described in previous examples to afford sodium7α-difluoromethyl-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 130 7-(2-Thienylacetamido)-7-aminocephalosporanic acid Step A:Benzhydryl 7,7-diazidocephalosporanate

Benzhydryl 7-bromo-7-azidocephalosporanate (10 mm.) is dissolved in 0.5M lithium azide (20 ml.) in dimethylformamide and heated at 68° C. forthree minutes under nitrogen. The solution is diluted with water (100ml.) and extracted with chloroform (20 ml.). The chloroform layer iswashed with two 60 ml. portions of water and dried over sodium sulfate.Evaporation of the solvent in vacuo yields crude benzhydryl7,7-diazidocephalosporanate.

Step B: Benzhydryl 7-amino-7-azidocephalosporanate

Benzhydryl 7,7-diazidocephalosporanate (10 mm.) are dissolved in ethylacetate and 3 g. 10% palladium-on-carbon are added. The mixture ishydrogenated at atmospheric pressure at 45° C. for one hour. There isthus obtained a crude isomeric mixture of benzhydryl7-amino-7-azidocephalosporanate.

Step C: Benzhydryl 7-(2-thienylacetamido)-7-Azidocephalosporanate

The isomeric mixture of benzhydryl 7-amino-7-azidocephalosporanates (5mm.) obtained in Step B is dissolved in methylene chloride (50 ml.) andcooled to 0° C. Collidine (5 mm.) and 2-thienylacetyl chloride (5mm.)are added and the solution is let stand at 0° C. for one hour. Themethylene chloride solution is washed with saturated sodium bicarbonatesolution, two 50 ml. portions of 1 N hydrochloric acid, a 1% sodiumbicarbonate solution (50 ml.) and water (50 ml.). The solvent is driedover sodium sulfate and evaporated to afford benzhydryl7-(2-thienylacetamido)-7-azidocephalosporanate in the form of a gum.

Step D: Benzhydryl 7-(2-thienylacetamido)-7-aminocephalosporanate

The benzhydryl 7-(2-thienylacetamido)-7-azidocephalosporanate (10 mm.)obtained in Step C is dissolved in ethyl acetate (50 ml.) and 3 g. of apalladium-on-charcoal catalyst is added. Hydrogenation is carried out atroom temperature at 40 psi for one hour. The catalyst is removed byfiltration and the solvent is evaporated in vacuo to afford crudebenzhydryl 7-(2-thienylacetamido)-7-aminocephalosporanate.

Step E: 7-(2-thienylacetamido)-7-aminocephalosporanic Acid

Benzhydryl 7-(2-thienylacetamido)-7-aminocephalosporanate is dissolvedin anisole (10 ml.) at 0° C. To this solution is added trifluoroaceticacid (15 ml.) cooled to 0° C. and the mixture is aged at roomtemperature for one hour. The excess trifluoroacetic acid-anisole isremoved by evaporation and the residue flushed twice with chloroform andevaporated to dryness. The crude product thus obtained is7-(2-thienylacetamido)-7-aminocephalosporanic acid.

EXAMPLE 131 7-(2-Thienylacetamido)-7-Azidocephalosporanic Acid

7-(2-Thienylacetamido)-7-azidocephalosporanate (1 mm.) is dissolved inanisole (10 ml.) at 0° C. To this solution is added trifluoroacetic acid(15 ml.) cooled to 0° C. and the mixture is aged at room temperature forone hour. Excess trifluoroacetic acid and anisole are removed byevaporation and the residue is flushed twice with chloroform andevaporated to dryness. The crude product thus obtained is7-(2-thienylacetamido)-7-azidocephalosporanic acid.

EXAMPLE 132 7-(2-Thienyl acetamido)-7-Guanidinocephalosporanic Acid

7-(2-Thienyl acetamido)-7-aminocephalosporanic acid (10 mm.) aredissolved in methanol (50 ml.) at room temperature.3,5-Dimethylamidinopyrazole (10 mm.) is added and the mixture is stirredfor 16 hours at room temperature. The solution is diluted with 100 ml.of water and extracted three times with ether. The ether layer is thendiscarded and the water layer is lyophilized to afford crude7-(2-thienyl acetamido)-7-guanidinocephalosporanic acid.

EXAMPLE 133 7-(2-Thienyl acetamido)-7-Ureidocephalosporanic Acid

7-(2-Thienyl acetamido)-7-aminocephalosporanic acid (10 mm.) aredissolved in dry pyridine (20 ml.) and the solution is cooled to -10° C.Carbamoyl chloride (10 mm.) dissolved in 20 ml. dry pyridine is addedslowly and the mixture is stirred for one hour at -10° C. and thenquenched with ice cold saturated sodium bicarbonate solution (100 ml.).The aqueous solution is extracted with ether to remove pyridine and thewater layer is lyophilized. The salt cake is slurried with methanol andfiltered and the methanol solution is evaporated to dryness to affordcrude 7-(2-thienyl acetamido)-7-ureidocephalosporanic acid.

EXAMPLE 134 7-(2-Thienylacetamido)-7-(N,N-Dimethylureido)cephalosporanic Acid

In a like manner N,N-dimethylcarbamoyl chloride is substituted forcarbamoyl chloride to give 7-(2-thienylacetamido)-7-(N,N-dimethylureido)cephalosporanic acid. Upon substitutingan equivalent amount of N,N-dimethylcarbamoyl chloride for the carbamoylchloride of Example 133 and following the procedure described thereinthere is thus obtained 7-(2-thienylacetamido)7(N,N-dimethylureido)cephalosporanic acid.

EXAMPLE 135 7-(2-Thienyl acetamido)-7-(N-Amidinoureido)cephalosporanicAcid

7-(2-Thienyl acetamido)-7-aminocephalosporanic acid (10 mm.) isdissolved in a solution of dimethylformamide (50 ml.) and pyridine (20ml.) and cooled to 0° C. The solution is treated with a 100 ml. solutionof toluene containing 17% phosgene (large excess). The reaction isallowed to stand for two hours and evaporated under vacuum to removetoluene and excess phosgene. The resulting solution containing7-(2-thienyl acetamido)-7-(chloroformamido)cephalosporanic acid is thencooled to 0° C. and guanidine (10 ml.) is added. The solution is aged at0° C. for two hours and quenched by the addition of a saturated sodiumbicarbonate solution. The mixture is extracted with ether to removepyridine and the water is removed by evaporation in vacuo and the saltcake extracted with dry methanol. Evaporation of the methanol yieldscrude 7-(2-thienyl acetamido)-7-(N-amidinoureido)cephalosporanic acid.

EXAMPLE 136 7-(2-Thienyl acetamido)-7-Sulfonamidocephalosporanic Acid

7-(2-Thienyl acetamido)-7-aminocephalosporanic acid (10 mmoles) isdissolved in pyridine (50 ml.) and cooled to 0° C. To this solution isadded pyridine-sulfur trioxide complex (10 mmoles). The mixture is agedfor three hours at 0° C. and the pyridine is removed in vacuo to affordcrude 7-(2-thienyl acetamido)-7-sulfonamidocephalosporanic acid.

EXAMPLE 137 7-(2-Thienyl acetamido)-7-Acetamidocephalosporanic Acid

7-(2-Thienyl acetamido)-7-aminocephalosporanic acid (1 mmole) isdissolved in 10 ml. of water at 0° C. containing one millimole of sodiumbicarbonate at 0° C. The mixture is treated with acetic anhydride (2mmoles), stirred at 0° C. for one hour and acidified with dilutehydrochloric acid (1 mmole). The precipitated product is filtered,washed with water and dried in vacuo to afford crude 7-(2-thienylacetamido)-7-acetamidocephalosporanic acid.

EXAMPLE 138 7-(2-Thienyl acetamido)-7-Methanesulfonamidocephalosporanicacid

Upon substituting an equivalent amount of methanesulfonyl chloride forthe acetic anhydride of Example 137 and otherwise following theprocedure described therein there is obtained 7-(2-thienylacetamido)-7-methanesulfonamidocephalosporanic acid.

EXAMPLE 1397-(2-Thienylacetamido)-7-(Ethoxycarbonylamino)-Cephalosporanic Acid StepA: Benzhydryl 7-azido-7-(ethoxycarbonylamino)cephalosporanate

Ethyl urethane (25.0 g.) is melted at 48° C. and benzhydryl7-bromo-7-azidocephalosporanate (5 mm.) is then added. To this solutionis also added in one portion molten ethyl urethane (15 g.) containingsilver tetrafluoroborate (15 mm.). The temperature is maintained at 50°C. for five minutes and the reaction is then quenched by adding to alarge volume of ether. The silver bromide formed is removed byfiltration through diatomaceous earth and the ether solution is washedsuccessively with water (400 ml.), a saturated sodium bicarbonatesolution (400 ml.) and two 400 ml. portions of water. The organic layeris dried with sodium sulfate, the solvent evaporated in vacuo and thegummy residue triturated several times with water to remove excess ethylurethane. There is thus obtained benzhydryl7-azido-7-(ethoxycarbonylamino)cephalosporanate.

Step B: Benzhydryl7-(2-thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanate

2-Thienylacetic anhydride (5 mm.), pyridine (0.1 ml.) and 10%palladium-on-carbon (3.0 g.) are added to a solution of benzhydryl7-azido-7-(ethoxycarbonylamino)cephalosporanate (5 mm.) dissolved inethyl acetate (25 ml.). The mixture is hydrogenated at room temperaturefor one hour. The catalyst is then removed by filtration and the residueevaporated to a glass under vacuum. There is thus obtained crudebenzhydryl7-(2-thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanate.

Step C: 7-(2-Thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanateacid

The benzhydryl7-(2-thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanate (1.0 mm.)obtained in Step B is dissolved in 10 ml. of anisole at 0° C. To thissolution is added trifluoroacetic acid (15 ml.) cooled to 0° C. and themixture is aged at room temperature for one hour. The excesstrifluoroacetic acid-anisole is removed by evaporation and the residueis flushed twice with chloroform and evaporated to dryness. The crudeproduct thus obtained is7-(2-thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanic acid.

EXAMPLE 140 7-(2-Thienylacetamido)-7-Methylaminocephalosporanic AcidStep A: Benzhydryl 7-azido-7-nitrocephalosporanate

Benzhydryl 7-bromo-7-azidocephalosporanate (10 mm.) is heated for fourminutes at 68° C. in dimethyl-formamide (60 ml.) containing lithiumnitrite (10 mm.). The solution is diluted with water (300 ml.) andextracted with two 50 ml. portions of chloroform. The chloroform layeris washed with three 100 ml. portions of water and dried over anhydroussodium sulfate. The crude product thus obtained is benzhydryl7-azido-7-nitrocephalosporanate.

Step B: Benzhydryl 7-(2-thienylacetamido)-7-nitrocephalosporanate

Benzhydryl 7-azido-7-nitrocephalosporanate (10 mm.) is dissolved inisopropanol (25 ml.) at 0° C. and to this solution sodium borohydride (5mm.) dissolved in isopropanol (25 ml.) is added slowly. The solution isaged for one hour at 0° C. and then quenched in ice water. The solutionis extracted with three 50 ml. portions of methylene chloride and theorganic layer is dried over sodium sulfate. To the dried methylenechloride solution is added ten millimoles of 2-thienylacetic anhydride(10 mm.) and collidine (10 mm.) at 0° C. After standing for 30 minutesat 0° C. the methylene chloride layer is extracted with two 50 ml.portions of 0.1 N hydrochloric acid and two 50 ml. portions of sodiumbicarbonate and, finally, with two 50 ml. portions of water. Themethylene chloride solution is then dried over sodium sulfate andevaporated in vacuo to afford a gum. The crude product thus obtained isbenzhydryl 7-(2-thienylacetamido)-7-nitrocephalosporanate.

Step C: 7-(2-Thienylacetamido)-7-nitrocephalosporanic acid

Upon substituting an equivalent amount of benzhydryl7-(2-thienylacetamido)-7-nitrocephalosporanate for the7-(2-thienylacetamido)-7-(ethoxycarbonylamino)cephalosporanate ofExample 139, Step C, and following the procedure described therein thereis thus obtained 7-(2-thienylacetamido)-7-nitrocephalosporanic acid.

Step D: 7-(2-Thienylacetamido)-7-aminocephalosporanic acid

7-(2-Thienylacetamido)-7-nitrocephalosporanic acid (10 mmoles) isreduced in methanol (50 ml.) using a palladium-on-charcoal catalyst (1.0g.) at 40 psi at room temperature for one hour. The catalyst is removedby filtration. The solvent is then removed by evaporation to affordcrude 7-(2-thienylacetamido)-7-aminocephalosporanic acid.

Step E: 7-(2-Thienylacetamido)-7-Methylaminocephalosporanic Acid

7-(2-Thienylacetamido)-7-aminocephalosporanic acid (10 mmoles) isdissolved in ethanol (100 ml.). To this solution is added platinum oxidecatalyst (0.1 g.) and formaldehyde (13 mm.). Hydrogenation isaccomplished at room temperature with two atmospheres hydrogen pressureover a 5 to 10 minute period. The catalyst is filtered off and thesolvent removed in vacuo to afford crude7-(2-thienylacetamido)-7-methylaminocephalosporanic acid.

EXAMPLE 141

By following the procedure described in Example 140 Steps A through E,all of the 7-amido-7-(alkylamino)cephalosporanate products of thisinvention may be obtained. Thus, for example, by substituting anequivalent amount of aralkanoic anhydride or heterocyclic anhydride forthe 2-thienylacetic anhydride recited in Step B and, upon substitutingan equivalent amount of alkaldehyde or aralkaldehyde for theformaldehyde recited in Step E therein, the corresponding7-amido-7-(N-alkylamino- and N-aralkylamino)cephalosporanic acids ofthis invention may be synthesized. The following equation andaccompanying Table illustrate this process and the products obtainedthereby: ##STR72##

                  TABLE I                                                         ______________________________________                                          R                     CH.sub.2 R.sup.1                                      ______________________________________                                                              CH.sub.3                                                 ##STR73##            C.sub.2 H.sub.5                                          ##STR74##            (CH.sub.2).sub.2 CH.sub.3                                ##STR75##                                                                                           ##STR76##                                               ##STR77##            CH(CH.sub.3).sub.2                                       ##STR78##                                                                                           ##STR79##                                               ##STR80##                                                                                           ##STR81##                                               ##STR82##            (CH.sub.2).sub.2 CH.sub.3                                ##STR83##                                                                                           ##STR84##                                              ______________________________________                                    

EXAMPLE 142 7-(2-Thienylacetamido)-7-(N,N-Dimethylamino)Cephalosporanicacid

7-(2-Thienylacetamido)-7-aminocephalosporanic acid (10 mm.) is dissolvedin acetic acid (100 ml.) containing formaldehyde (40 mm.). To thissolution is added platinum oxide catalyst (0.2 g.) and the hydrogenationis carried out at 4-6 atmospheres of hydrogen at 35° C. for eight hours.The catalyst is then filtered off and the solvent is removed byevaporation in vacuo to afford crude7-(2-thienylacetamido)-7-(N,N-dimethylamino)cephalosporanic acid.

EXAMPLE 143 7-Phenylacetamido-7-(Ethoxycarbonylamino)cephalosporanicAcid Step A: Benzhydryl7-phenylacetamido-7-(ethoxycarbonylamino)cephalosporanate

Phenylacetic anhydride (5 mm.), pyridine (0.1 ml.) and Bolhoffercatalyst (3.0 g.) are added to a solution of benzhydryl7-azido-7-(ethoxycarbonylamino)cephalosporanate (5 mm.) dissolved inethyl acetate (25 ml.). The mixture is hydrogenated at room temperaturefor one hour. The catalyst is then removed by filtration and the residueevaporated under vacuum to afford crude benzhydryl7-phenylacetamido-7-(ethoxy-carbonylamino)cephalosporanate.

Step B: 7-Phenylacetamido-7-(ethoxycarbonylamino)cephalosporanic acid

Benzhydryl 7-phenylacetamido-7-(ethoxycarbonylamino)cephalosporanate(1.0 mm.) is dissolved in anisole (10 ml.) at 0° C. and to this solutionis added trifluoroacetic acid (15 ml.) cooled to 0° C. The mixture isthen aged at room temperature for one hour. Excess trifluoroaceticacid-anisole is removed by evaporation and the residue is flushed twicewith chloroform and evaporated to dryness to afford crude7-phenylacetamido-7-(ethoxycarbonylamino)cephalosporanic acid.

EXAMPLE 144

By following the procedure described in Example 142 for the preparationof 7-(2-thienylacetamido)-7-(N,N-dimethylamino)cephalosporanic acid allof the 7-amido-7-(N,N-dialkylamino)cephalosporanic acids of thisinvention may be obtained. Thus, for example, by substituting anequivalent amount of alkaldehyde for the formaldehyde recited in Example142 and following the procedure described therein, the corresponding7-(2-thienylacetamido)-7-(N,N-dialkylamino)cephalosporanic acid productsmay be obtained. The following equation and Table II illustrate thisprocess and the products obtained thereby: ##STR85##

                  TABLE II                                                        ______________________________________                                         R.sup.2            OCHR.sup.2                                                ______________________________________                                         C.sub.2 H.sub.5    OCHCH.sub.3                                               (CH.sub.2).sub.2 CH.sub.3                                                                         OCHCH.sub.2 CH.sub.3                                                           ##STR86##                                                 ##STR87##                                                                                         ##STR88##                                                (CH.sub.2).sub.3 CH.sub.3                                                                         OCH(CH.sub.2).sub.2 CH.sub.3                              (CH.sub.2).sub.4 CH.sub.3                                                                         OCH(CH.sub.2).sub.3 CH.sub.3                              ______________________________________                                    

EXAMPLE 145 Sodium7-Ethoxycarbonylamino-7-(2-Thienylacetamido)cephalosporanate Step A:Benzhydryl 7-Azido-7-(Ethoxycarbonylamino)cephalosporanatecephalosporanate

Benzhydryl 7-azido-7-bromocephalosporanate (3.9 g.) is added toethylcarbamate (36 g.) maintained at 65° C. To the resulting mixture isadded portionwise as a melt silver tetrafluoroborate (3.6 g.) dissolvedin ethylcarbamate (18 g.) and the reaction mixture is maintained at67°-70° C. for five minutes. The mixture is then poured into ether withstirring and the resulting slurry is filtered through celite to removethe silver bromide. The ether is extracted successively with water (100ml.) saturated aqueous sodium bicarbonate (100 ml.) solution (100 ml.)and two portions of water (100 ml.). The extracted ether solution isdried over sodium sulfate and then evaporated under diminished pressure.The resulting residue is triturated three times with a small amount ofwater and then dissolved in chloroform. The chloroform solution is driedover sodium sulfate and evaporated to dryness to afford 2.1 g. ofbenzhydryl 7-azido-7-(ethoxycarbonylamino)cephalosporanate.

Step B: Benzhydryl 7-Amino-7-(Ethoxycarbonylamino)cephalosporanate

Benzhydryl 7-azido-7-(ethoxycarbonylamino)cephalosporanate (1.0 g.) isdissolved in dioxane (100 ml.). Platinum oxide (1.0 g.) is added and thereaction mixture is stirred under hydrogen at atmospheric pressure forone hour. Another 1.0 g. quantity of platinum oxide is added and thereaction mixture is again placed under hydrogen and stirred for threehours until the azide is completely reacted as determined by an infraredanalysis of aliquots. The solvent is removed under reduced pressure andthe residue taken up in chloroform (50 ml.) and filtered through silicagel G in chloroform in a 60 ml. sintered glass funnel. The resultingmaterial is eluted with chlorofrom until 200 ml. of chloroform has beencollected. The chloroform is then removed under reduced pressure toafford benzhydryl 7-amino-7-(ethoxycarbonylamino)cephalosporanate (0.6g.).

Step C: Benzhydryl7-Ethoxycarbonylamino-7-(2-Thienylacetamido)cephalosporanate

The benzhydryl 7-amino-7-(ethoxycarbonylamino)cephalosporanate (0.6 g.)obtained in Step B is taken up in methylene chloride (25 ml.) and cooledto 0° C. 2-Thienylacetyl chloride (0.6 ml., 0.038 mole) is addeddropwise over 30 seconds followed by the addition of pyridine (0.6 ml.,0.01 mole) 60 seconds later. The reaction mixture is stirred at 0° C.for 15 minutes and poured into crushed ice. The mixture is agitated andthe organic layer separated and washed once with water (20 ml.), oncewith 5% sodium bicarbonate (20 ml.) and once again with water (20 ml.).The methylene chloride mixture is then dried and evaporated to drynessto afford 1.42 g. of crude benzhydryl7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate. Thismaterial is placed on a column of 60 g. of silica gel under benzene andthe column is eluted with benzene, taking 100 ml. fractions followed by300 ml. of methylene chloride benzene (1:1) in three fractions and 500ml. of methylene chloride in five fractions. The product is removed fromthe column by eluting with chloroform (400 ml.) in four fractions,affording 0.55 g. of benzhydryl7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate. Thismaterial is taken up in methylene chloride (25 ml.) and stirred at roomtemperature with 20 ml. of a solution of sodium bicarbonate (0.120 g.)in water for 0.5 hour. The resulting layers are then separated and theorganic layer is washed with water, dried and evaporated to dryness toafford 0.4 g. of benzhydryl7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate.

Step D. Sodium7-Ethoxycarbonylamino-7-(2-Thienylacetamido)cephalosporanate

Benzhydryl 7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate(0.4 g.) is dissolved in anisole (2.5 ml.) and treated withtrifluoroacetic acid (10 ml.) at room temperature for 10 minutes. Thetrifluoroacetic acid and anisole are removed under reduced pressurewhile maintaining the temperature below 40° C., and the residue is takenup in chloroform (25 ml.) and treated with 20 ml. of water containing0.120 g. of sodium bicarbonate. The mixture is stirred for 0.5 hour atroom temperature and the organic phase is separated and washed withwater. The combined aqueous phase is then washed twice with methylenechloride and lyophilized to afford 0.32 g. of sodium7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate as abrownish solid.

EXAMPLE 1467-Ethoxycarbonylamino-7-(2-Thienylacetamido)-3-PyridiniumdecephalosporanicAcid

Sodium 7-ethoxycarbonylamino-7-(2-thienylacetamido)cephalosporanate (1.3g.) is dissolved in 3 cc. of a buffer solution made up by dissolvingpotassium iodide (30 g.) in water (20 ml.) containing 5 ml. of pyridineand adjusting the pH to 6.5 with dilute acetic acid. The resultingreaction mixture is stirred at 80° C. for two hours and then cooled toroom temperature. The solution is then freeze dried, dissolved in 25%acetic acid (40 ml.), filtered and the filtered solution subjected toelectrophoresis to obtain7-ethoxycarbonylamino-7-(2-thienylacetamido)-3-pyridiniumdecephalosporanicacid.

EXAMPLE 147 Sodium7-ethoxycarbonylamino-7-phenylacetamidocephalosporanate

When benzhydryl 7-amino-7-ethoxycarbonylaminocephalosporanate is reactedwith phenylacetyl chloride in place of thienylacetyl chloride asdescribed in Example 145 above, benzhydryl7-ethoxycarbonylamino-7-phenylacetamidocephalosporanate is obtained.Using the procedures described in Example 145, this ester is convertedto the sodium salt.

EXAMPLE 148 Sodium7-ethoxycarbonylamino-7-thiophenoxyacetamido-cephalosporanate

When benzhydryl 7-amino-7-ethoxycarbonylaminocephalosporanate is reactedwith an equivalent amount of phenylthioacetyl chloride in place ofthienylacetyl chloride as described in Example 145 above, benzhydryl7-ethoxycarbonylamino-7-thiophenoxyacetamidocephalosporanate isobtained. Using the procedures described in Example 145 above, thisester is converted to the sodium salt.

EXAMPLE 149 Sodium7-ethoxycarbonylamino-7-(2-furylacetamido)cephalosporanate

The sodium salt of7-ethoxycarbonylamino-7-(2-furylacetamido)cephalosporanic acid isprepared by reacting the 7-amino-7-ethoxycarbonylaminocephalosporanateester with furylacetyl chloride and then converting the ester to thesodium salt as described in Example 145.

EXAMPLE 150 Sodium 7-amino-7-(2-thienylacetamido)cephalosporanate

When benzhydryl 7-azido-7-bromocephalosporanate is reacted witht-butylcarbamate in place of ethylcarbamate as described in Example 145above, the 7-t-butylcarbonylamino-7-aminocephalosporanate ester isobtained. Treatment of this product as described in Example 145 aboveaffords sodium 7-amino-7-(2-thienylacetamido)cephalosporanate.

EXAMPLE 151 Sodium 7-hydrazino-7-(2-thienylacetamido)cephalosporanate

When benzhydryl 7-azido-7-bromocephalosporanate is reacted witht-butylcarbazate in place of ethylcarbamate as described in Example 145above, the resulting product is reacted as described in Example 145 andsodium 7-hydrazino-7-(2-thienylacetamido)cephalosporanate is obtained.

EXAMPLE 152 Sodium 7-azido-7-(2-thienylacetamido)cephalosporanate StepA: Benzhydryl 7-azido-7-(2-thienylacetamido)cephalosporanate

Benzhydryl 7-azido-7-bromocephalosporanate (3.6 g.) is added to2-thienylacetamide (30 g.) and the mixture is warmed to 65°-70° C. Tothis reaction mixture is added silver tetrafluoroborate (3.6 g.) and2-thienylacetamide (15 g.) and the mixture heated to 65°-70° C. for 10minutes. The reaction mixture is poured into ether (200 ml.) withstirring and the resulting slurry is filtered through diatomaceous earthto remove the silver bromide. The ether solution is extracted with water(100 ml.), saturated aqueous sodium bicarbonate solution (100 ml.) andtwo 100 ml. portions of water. The ether layer is then dried over sodiumsulfate and evaporated under diminished pressure. The resulting residueis triturated twice with a small amount of water and then dissolved inchloroform (25 ml.). After drying the chloroform solution over sodiumsulfate, it is evaporated under diminished pressure to afford 2.4 g. ofbenzhydryl 7-azido-7-(2-thienylacetamido)cephalosporanate.

Step B: Sodium 7-azido-7-(2-thienylacetamido)cephalosporanate

The benzhydryl 7-azido-7-(2-thienylacetamido)cephalosporanate obtainedin Step B is deblocked by reaction with trifluoroacetic acid and anisoleand the product recovered as the sodium salt following the proceduredescribed in Example 145.

EXAMPLE 153

When the process of Example 130, step A is carried out starting withbenzhydryl 7-chloro-7-azidocephalosporanate using an equivalent amountof potassium azide or sodium azide in place of the lithium azide,benzhydryl-7,7-diazocephalosporanate is obtained. Reduction of thisproduct, acylation of the 7-amino compound with phenylacetyl chlorideand furylacetyl chloride, and cleavage of the benzhydryl group followingthe procedures described in the foregoing examples affords7-benzylacetamido-7-aminocephalosporanic acid and7-(2-furylacetamido)-7-aminocephalosporanic acid, respectively.

EXAMPLE 154 Sodium 7-Bromo-7-Phenylacetamidocephalosporanate Step A:Benzhydryl 7-Aminocephalosporanate

P-toluenesulfonic acid monohydrate (4.3 g., 0.22 mole) is added withstirring at room temperature, to a slurry of 7-aminocephalosporanic acid(6.8 g., 0.25 mole) in peroxide-free dioxane (300 ml.). The clearsolution is concentrated in vacuo and flushed twice with dioxane. Theresidue is then dissolved in dioxane (300 ml.) at room temperature and asolution of diphenyldiazomethane (10 g., 0.05 mole) in dioxane (25 ml.)is added dropwise over 15 minutes. The resulting wine-colored solutionis stirred for an additional 30 minutes and methanol (25 ml.) is thenadded to destroy the excess diphenyldiazomethane. The mixture isconcentrated in vacuo and the residue is layered between methylenechloride (200 ml.) and water (200 ml.) containing dipotassium phosphate(10 g., pH 8.5). The organic phase is washed with water, dried oversodium sulfate and concentrated in vacuo to yield an oil. The oil isstirred with ether (100 ml.) for one hour and the resulting precipitateis filtered, washed with ether and dried to constant weight 4.7 g. (43%yield). The compound thus obtained is benzhydryl7-aminocephalosporanate, m.p. 126°-128° C.

Analysis: Calc.: C, 63.0; H, 5.01; N, 6.37. Found: C, 62.7; H, 5.18; N,5.18.

Infrared in chloroform: 5.6μ (B-lactam C═O) and 5.8μ (ester C═O).

Nuclear Magnetic Resonance in deuterated chloroform: ##STR89##

Step B: Benzhydryl 7-Diazocephalosporanate

To a mixture of sodium nitrite (1.6 g.), water (30 ml.) and methylenechloride (40 ml.) is added benzhydryl 7-aminocephalosporanate (880 mg.,0.002 mole) with stirring at 0° C. A solution of para-tolueuesulfonicacid (760 mg., 0.004 mole) in water (5 ml.) is then added over a fewminutes and the mixture is stirred at 0° C. for 20 minutes. The organicphase is then cut away, washed with ice water (10 cc.), dried oversodium sulfate at 0° C., filtered and concentrated in vacuo at roomtemperature to yield 900 mg. of benzhydryl 7-diazocephalosporanate inthe form of a glass. Infrared is 4.8μ (strong N═N), 5.6μ (β-lactam C═O)and 5.8μ (ester C═O).

Nuclear Magnetic Resonance in deuterated chloroform: ##STR90##

Step C: Benzhydryl 7-Bromo-7-Azidocephalosporanate Preparation ofTriethylammonium Azide

To a slurry of sodium azide (1.5 g.) in water (5 ml.) and methylenechloride (10 ml.) maintained at -10° C. is added 50% sulfuric acid (4ml.) dropwise over a five minute period. The mixture is stirred anadditional five minutes and the organic phase is poured off from theaqueous paste and the aqueous extract is washed with methylene chloride(5 cc.). The combined organic phase is dried over calcium chloride andthe decanted azide solution is brought to pH 7 with triethylamine. Thetriethylammonium azide thus obtained is stored at -10° C.

Preparation of Bromineazide

To methylene chloride (8 ml.) maintained at 0° C. is added sodium azide(2.66 g., 0.04 mole) followed by the addition of bromine (0.65 g.,0.0042 mole). To this mixture is added dropwise, with stirring, at 0° C.concentrated hydrochloric acid (2 ml.) and stirring is continued for anadditional three hours at 0° C. The organic layer is decanted and theaqueous layer is extracted with methylene chloride (5 ml.). Theextracted bromineazide is stored at -10° C.

To a solution of benzhydryl 7-diazocephalosporanate (900 mg.) inmethylene chloride (20 ml.) and nitro methane (10 ml.) maintained at0°-10° C. is added the triethylammonium azide solution (as preparedabove) followed by the addition of the bromineazide solution (asprepared above). Water (50 ml.) is then added followed by the additionof sufficient solid sodium bicarbonate to bring the pH of the solutionto 8. The organic layer is separated and extracted with two 20 ml.portions of water, dried over sodium sulfate and concentrated in vacuoto yield 900 mg. (83%) of benzhydryl 7-bromo-7-azidocephalosporanate.

Thin layer chromatography on silica gel with chloroform shows a majorspot for this product at R_(f) 0.2. Chromatography of the 900 mg. ofproduct on 25 gms. of silica gel with chloroform gives 400 mg. (39%)single spot material as an oil.

Infrared in chloroform: 4.7μ (N₃), 5.56μ (β-lactam C═O) and 5.75μ (esterC═O).

Nuclear Magnetic Reasonance: in deuterated chloroform: ##STR91##

Step D: Benzhydryl 7-Bromo-7-Phenylacetamidocephalosporanate

Sodium borohydride (0.1 g.) is added to a solution of benzhydryl7-azido-7-bromocephalosporanate (0.5 g.) in bis-(2-methoxyethyl)ether(10 ml.) and the resulting mixture is stirred overnight under nitrogen.The mixture is then poured into ice water, adjusted to pH 8, extractedwith methylene chloride, dried and evaporated to afford benzhydryl7-amino-7-bromocephalosporanate.

The benzhydryl 7-amino-7-bromocephalosporanate is then treated with twoequivalents of phenylacetic acid anhydride in methylene chloridesolution and the resulting mixture is stirred for 0.5 hour and thenpoured into ice cold water. The resulting aqueous solution is extractedinto methylene chloride and the solvent is dried over sodium sulfate andevaporated under reduced pressure to obtain benzhydryl7-bromo-7-phenylacetamidocephalosporanate. This product is purifiedfurther by chromatography over silica gel to obtain pure benzhydryl7-bromo-7-phenylacetamidocephalosporanate.

Step E: Sodium 7-Bromo-7-Phenylacetamidocephalosporanate

Benzhydryl 7-bromo-7-phenylacetamidocephalosporanate (0.4 g.) isdissolved in anisole (3.5 ml.) and treated with trifluoroacetic acid (10ml.) at room temperature for 10 minutes. The trifluoroacetic acid andanisole are removed under reduced pressure while maintaining thetemperature below 40° C. The residue is then taken up in chloroform (25ml.) and treated with water (20 ml.) containing sodium bicarbonate(0.120 g.). The resulting mixture is stirred for 0.5 hour at roomtemperature and the organic phase is separated and washed with water.The combined aqueous phase is then washed twice with methylene chlorideand lyophilized to afford sodium7-bromo-7-phenylacetamidocephalosporanate as a brownish solid.

EXAMPLE 155 Sodium-7-Chloro-7-(2-Thienylacetamido)cephalosporanate StepA: Benzhydryl 7-Azido-7-Chlorocephalosporanate

To a solution of benzhydryl 7-diazocephalosporanate (900 mg.) inmethylene chloride (20 ml.) and nitromethane (10 ml.) at 0°-10° C. isadded triethylammonium azide (prepared as described in Example 154, StepC) followed by the addition of the chlorineazide (prepared as describedbelow). To this reaction mixture is added 50 ml. of water and sufficientsolid sodium bicarbonate to bring the pH of the mixture to 8. Theorganic layer is separated and extracted with two 20 ml. portions ofwater, dried over sodium sulfate and concentrated under reduced pressureto yield benzhydryl 7-azido-7-chlorocephhalosporanate.

Preparation of Chlorineazide

A 5.25% solution (100 ml.) of sodium hypochlorite in water is mixed withchlorine (50 ml.) and cooled to -10° C. To this mixture is added sodiumazide (4.3 g.) followed by the addition of aqueous acetic acid (10 ml.)prepared by diluting 4 ml. of glacial acetic acid to 10 ml. The additionof the acetic acid is made over a period of 15 minutes and the reactionmixture is stirred for an additional 15 minutes at -10° C. The aqueousphase is then separated, frozen by cooling in a solid carbon dioxideacetone bath and the organic phase is decanted and dried over anhydroussodium sulfate. This methylene chloride solution is then used in thereaction described above.

Step B: Sodium 7-Chloro-7-(2-Thienylacetamido)cephalosporanate

The benzhydryl 7-azido-7-chlorocephalosporanate obtained in Step A isreduced with sodium borohydride as in Example 154, Step D to obtainbenzhydryl 7-amino-7-chlorocephalosporanate and this product is thenacylated by reaction with 2-thienylacetic acid anhydride to affordbenzhydryl 7-chloro-7-(2-thienylacetamido)-cephalosporanate. Uponsubstituting benzhydryl 7-chloro-7-(2-thienylacetamido) cephalosporanatefor the benzhydryl 7-bromo-7-phenylacetamidocephalosporanate of Example154, Step E, and following the procedure described therein there is thusobtained sodium-7-chloro-7-(2-thienylacetamido)-cephalosporanate.

EXAMPLE 156 Sodium 7-Fluoro-7-(2-Furylacetamido)cephalosporanate Step A:Benzhydryl 7-Azido-7-Fluorocephalosporanate

To a solution of benzhydryl 7-azido-7-bromocephalosporanate (0.5 g.) inanhydrous acetonitrile (50 ml.) is added tetraethylammonium fluoride(3.0 g.). The resulting mixture is stirred overnight at room temperatureand then poured into water and extracted into methylene chloride toafford benzhydryl 7-azido-7-fluorocephalosporanate.

Step B: Sodium 7-Fluoro-7-(2-Furylacetamido)cephalosporanate

The benzhydryl 7-azido-7-fluorocephalosporanate obtained in Step A isreduced with sodium borohydride to obtain benzhydryl7-amino-7-fluorocephalosporanate and this product is then acylated byreaction with 2-furylacetic acid anhydride and the resulting benzhydryl7-fluoro-7-(2-furylacetamido)-cephalosporanate is converted to thesodium salt following the procedure described in Example 154, Step E.

EXAMPLE 157 Sodium 7-(2-Thienylacetamido)-7-Chlorocephalosporanate StepA: Benzhydryl 7-Aminocephalosporanate

7-Aminocephalosporanic acid (272 mg.) is slurried for five minutes at250° C. in dioxane (7 ml.) with p-toluenesulfonic acid monohydrate (170mg.). Methanol (2 ml.) is added, the solvents are removed under vacuumand dioxane is twice added and evaporated in vacuo. Dioxane (8 ml.) isthen added followed by the addition of diphenyldiazomethane (290 mg.).After the evolution of nitrogen is complete, the solvent is distilledunder vacuum and the residue is stirred with methylene chloride (10 ml.)and water (10 ml.) containing sufficient dibasic potassium phosphate tobring the pH of the mixture to 8. The layers are separated and theaqueous portion is extracted twice more with methylene chloride. Thecombined organic layers are dried with sodium sulfate, filtered and thenevaporated to yield an oily crystalline material. Washing with etheraffords 150 mg. (35% yield) of the benzhydryl 7-aminocephalosporanate inthe form of a dry solid, m.p. 110°-115° C.

Step B: Benzhydryl 7-(p-Nitrobenzylideneamino)cephalosporanate

Benzhydryl 7-aminocephalosporanate (438 mg.) is refluxed for one hour inbenzene (50 ml.) with p-nitrobenzaldehyde (151 mg.) in an azeotropicdrying apparatus. The solvent is then eliminated by vacuum distillationto afford 571 mg. of crude benzhydryl7-(p-nitrobenzylideneamino)cephalosporanate. This compound may be useddirectly in the next step or, if desired, may be purified byrecrystallization from a mixture of one part benzene and two partscyclohexane.

Step C: Benzhydryl 7-(p-Nitrobenzylideneamino)-7-Chlorocephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate (571 mg.) isstirred at 0° C. under nitrogen in dimethylformamide (5 ml.). Acatalytic amount of potassium t-butoxide is added and then t-butylhypochlorite (109 mg.) is added with rapid stirring over a one to twominute period.

The solvent is evaporated in vacuo and the resulting oily residue istaken up in benzene (25 ml.), washed four times with water (10 ml.),dried with magnesium sulfate and then filtered and evaporated to affordthe benzhydryl 7-(p-nitrobenzylideneamino)-7-chlorocephalosporanate. Thecrude material thus obtained may be used directly in the next step or,if desired, may be purified by chromatography on neutral silica gel andelution with 2% ethyl acetate in chloroform.

Step D: Benzhydryl 7-Amino-7-Chlorocephalosporanate Hydrochloride

Benzhydryl 7-(p-nitrobenzylideneamino)-7-chlorocephalosporanate (606mg.) and aniline hydrochloride (260 mg.) are stirred together for onehour at 25° C. in methanol (10 ml.). The methanol is removed at 0.1 mm.pressure and 30° C. and the residue is then covered with ether andallowed to stand for one hour to effect crystallization. The solid istriturated with ether, filtered and then washed with ether to afford thebenzhydryl 7-amino-7-chlorocephalosporanate hydrochloride. This materialcontains some unreacted aniline hydrochloride but is of sufficientpurity as to be useable directly (i.e., without further purification) inthe next step.

Step E: Benzhydryl 7-(2-Thienylacetamido)-7-Chlorocephalosporanate

Benzhydryl 7-amino-7-chlorocephalosporanate hydrochloride, obtained instep D, is added to methylene chloride (25 ml.) and this mixture issitrred vigorously at -10° C. Triethylamine (0.5 g.) is added slowlywhereupon the free amine, i.e., the benzhydryl7-amino-7-chlorocephalosporanate is obtained. 2-Thienylacetyl chloride(0.5 g.) is then added and the reaction mixture is allowed to warm toroom temperature. Excess acid chloride is then hydrolyzed by shakingwith water and the methylene chloride layer is washed successively witha dilute phosphoric acid solution buffered to pH 2, water and dilutesodium bicarbonate solution. After drying with magnesium sulfate thesolution is filtered and evaporated to afford a mixture of benzhydryl7-(2-thienylacetamido)-7-chlorocephalosporanate andN-phenyl-2-theinylacetamide. Pure benzhydryl7-(2-thienylacetamido)-7-chlorocephalosporanate is obtained via silicagel chromatography by eluting the said mixture with four partschloroform and one part ethyl acetate.

Step F: Sodium 7-(2-Thienylacetamido)-7-Chlorocephalosporanate

Benzhydryl 7-(2-thienylacetamido)-7-chlorocephalosporanate (597 mg.) isdissolved in anisole (0.8 g.) and cooled to 0° C. Trifluoroacetic acid(4 ml.) precooled to 0° C. is added and the reaction is allowed toproceed for two minutes at 0° C. A vacuum of 0.1 mm. of mercury isimmediately applied and the reaction mixture is allowed to warm to roomtemperature without external heating. Anisole is then pushed over at 30°C. at 0.1 mm of mercury and a few ml. of anisole is added to the residueand pulled off at 30° C. at 0.1 mm. of mercury to insure total removalof trifluoroacetic acid. The product is treated with water (5 ml.)containing sodium bicarbonate (84 mg.) and then lyophilized. Theresulting powder is washed thoroughly with ether and dried to affordpure sodium 7-(2-thienylacetamido)-7-chlorocephalosporanate.

EXAMPLE 158 Sodium 7-(2-Thienylacetamido)-7-Bromocephalosporanate StepA: Benzhydryl 7-(2-Thienylacetamido)-7-Bromocephalosporanate

Benzhydryl 7-bromo-7-azidocephalosporanate (543 mg.) is added to2-thienylacetic anhydride (500 mg.) and a platinum oxide catalyst (1.0g.) in dioxane (50 ml.) and the said mixture is hydrogenated atatmospheric pressure for four hours. The resulting mixture is filteredto remove the platinum oxide catalyst, water (0.5 ml.) is added and thesolution is allowed to stand for 15 minutes to effect an hydrolysis ofany unreacted 2-thienylacetic anhydride. The solution is then distilledunder vacuum and the crude benzhydryl ester of7-(2-thienylacetamido)-7-bromocephalosporanate thus obtained isdissolved in ether (25 ml.), washed with aqueous sodium bicarbonate,dried over magnesium sulfate, filtered and evaporated. The product thusobtained is benzhydryl 7-(2-thienylacetamido)-7-bromocephalosporanatewhich, if desired, may be purified further by chromatography on silicagel by eluting with four parts chloroform and one part ethyl acetate.

Step B: Sodium 7-(2-Thienylacetamido)-7-Bromocephalosporanate

Benzyhydryl 7-(2-thienylacetamido)-7-bromocephalosporanate (641 mg.) isdissolved in anisole (0.8 mole) and cooled to 0° C. Trifluoroacetic acid(4 ml.) precooled to 0° C. is added and the mixture is allowed toproceed for two minutes at 0° C. The reaction mixture is then placedimmediately under vacuum and allowed to warm to room temperature withoutexternal heating. The mixture is then subjected to vacuum distillationwhereupon anisole distills over at 30° C./0.1 mm. mercury. The productthus obtained is treated with water (5 ml.) containing sodiumbicarbonate (84 mg.) and lyphilized to a powder; this powder is washedthoroughly with ether and dried to afford sodium7-(2-thienylacetamido)-7-bromocephalosporanate.

EXAMPLE 159 Sodium 7-(2-thienylacetamido)-7-chlorocephalosporanate StepA: Benzhydryl 7-(2-thienylacetamido)-7-chlorocephalosporanate

By substituting benzhydryl 7-chloro-7-azidocephalosporanate (499 mg.)for the benzhydryl 7-bromo-7-chlorocephalosporanate recited in Example158 and following the procedure described therein, there is thusobtained the benzhydryl 7-(2-thienylacetamido)-7-chlorocephalosporanate.

Step B: Sodium 7-(2-thienylacetamido)-7-chlorocephalosporanate

By substituting benzhydryl7-(2-thienylacetamido)-7-chlorocephalosporanate for the benzhydryl7-(2-thienylacetamido)-7-bromocephalosporanate in Example 158, Step Band following the procedure described therein, there is thus obtainedthe sodium 7-(2-thienylacetamido)-7-chlorocephalosporanate.

EXAMPLE 160 Sodium7-[α-(α-Aminophenyl(Acetamido]-7-(Dimethylphosphono)cephalosporanateStep A: Benzhydryl 7-Azido-7-(Dimethylphosphono)Cephalosporanate

To a solution of benzhydryl 7-azido-7-bromocephalosporanate (272 mg.,0.0005 mole) in dimethoxyethane (10 ml.) is added silverdimethylphosphite (120 mg., 0.00055 mole) (prepared by mixing equimolaramounts of dimethylphosphite and silver oxide in water and filtering thesolid which slowly forms). The mixture is stirred overnight at roomtemperature protected from light, air, and moisture. The inorganicmaterial is filtered and the filtrate is concentrated at a lowtemperature and pressure. The residue is dissolved in methylene chlorideand the solution is washed with a cold, dilute, aqueous solution ofsodium bicarbonate. It is dried and concentrated at reduced pressure.The residue is re-dissolved in a small amount of chloroform and themixture is purified by chromatography on silica gel using chloroform asthe elution solvent. The initial eluate contains a little recoveredstarting material. The second elution fraction is concentrated atreduced pressure to afford benzhydryl7-azido-7-(dimethylphosphono)cephalosporanate.

Step B: Benzhydryl 7-Amino-7-(Dimethylphosphono)Cephalosporanate

Benzhydryl 7-azido-7-(dimethylphosphono)cephalosporanate (200 mg.) isdissolved in dioxane (20 ml.) and hydrogenated at room temperature andnormal pressure for one hour using platinium oxide (200 mg.) ascatalyst. A fresh 200 mg.-portion of catalyst is added and thehydrogenation is continued for an additional two hours. The solvent isremoved at low temperature and pressure. The dry residue is dissolved inchloroform and the solution is filtered by suction through a layer ofsilica gel to remove the suspended, spent catalyst. The silica gel iswashed copiously with chloroform to elute any adsorbed material. Thefiltrate is concentrated at reduced pressure to afford benzhydryl7-amino-7-(dimethylphosphono)cephalosporanate suitable for use in thenext step without further purification.

Step C: Benzhydryl7-α-(α-Aminophenyl)Acetamido-7-(Dimethylphosphono)Cephalosporanate

A solution of benzhydryl 7-amino-7-(dimethylphosphono)cephalosporanate(273 mg.) in methylene chloride (15 ml.) is stirred at 0° C. andα-amino-α-phenylacetyl chloride hydrochloride (220 mg.) is added.Immediately thereafter pyridine (0.5 ml.) is added, and the mixture isstirred at 0° C. for 15 minutes and then poured onto crushed icecontaining a slight excess of hydrochloric acid. The layers areseparated and the organic phase is washed with an excess of a cold,dilute, aqueous solution of sodium bicarbonate. It is dried andconcentrated yielding benzhydryl7-β-(α-aminophenyl)acetamido-7-(dimethylphosphono)cephalosporanate. Thecrude material is purified by chromatography on silica gel withchloroform as the elution agent. A small amount of the isomer,benzhydryl7-α-(α-aminophenyl)acetamido-7-(dimethylphosphono)cephalosporanate, isalso isolated from the chromatographed mixture.

Step D: Sodium7-β-(α-Aminophenyl)Acetamido-7-(Dimethylphsophono)Cephalosporanate

A solution of benzhydryl7-β-(α-aminophenyl)acetamido-7-(dimethylphosphono)cephalosporanate (70mg.) and anisole (11 mg.) in methylene chloride (0.5 ml.) is stirred inan ice bath. A cold solution of trifluoroacetic acid (23 mg.) inmethylene chloride (0.2 ml.) is added in increments over a period offive minutes. After an additional 30 minutes the mixture is concentratedby means of a high vacuum pump. The residue is taken up in benzene (5ml.) and 3 ml. of an aqueous solution of sodium bicarbonate (8.4 ml.) isadded. The mixture is then stirred well at room temperature for 15minutes, the layers are separated and the organic phase is stirred oncemore with water (2 ml.), after which the layers are again separated. Thecombined aqueous extracts are washed once with ether and are lyophilizedto yield sodium7-β-(α-aminophenyl)acetamido-7-(dimethylphosphono)cephalosporanate.

EXAMPLE 161 Sodium7-[(Dimethylamino)Methoxyphosphinyl]-7-(α-2-Furylacetamido)CephalosporanateStep A: Benzhydryl7-Azido-7-[(Dimethylamino)Methoxyphosphinyl]Cephalosporanate

A mixture of benzhydryl 7-azido-7-bromocephalosporanate (545 mg.),N,N,O-trimethylphosphonamidic acid (246 mg.) and silvertetrafluoroborate (195 mg.) in dimethoxyethane (5 ml.) is stirredovernight at room temperature protected from air, light, and moisture.The solution is filtered from inorganic salts and concentrated todryness at low temperature and pressure. The residue is dissolved inmethylene chloride and the solution is washed with cold, aqueous sodiumbicarbonate solution, then with water and is dried and concentrated. Theresidue is redissolved in chloroform and the solution is charged onto acolumn of silica gel suspended in chloroform. Elution of the column withchloroform and concentration of the eluate yields benzhydryl7-azido-7-[(dimethylamino)methoxyphosphinyl]cephalosporanate.

Step B: Benzhydryl7-Amino-7-[(Dimethylamino)Methoxyphosphinyl]Cephalosporanate

Benzhydryl 7-azido-7-[(dimethylamino)methoxyphosphinyl]cephalosporanate(250 mg.) in dioxane (25 ml.) is hydrogenated at room temperature andunder normal pressure using a platinum oxide catalyst (500 mg.) added intwo equal portions with an interval of one hour. The hydrogenation iscontinued for 2-3 hours after the second addition, or until the azide isabsent as determined by infrared spectroscopy. The solvent is completelyremoved at low temperature and pressure. The residue is dissolved inchloroform and the spent catalyst is removed by filtration through alayer of silica gel, which adsorbent is thoroughly washed withchloroform. The filtrate is then concentrated at reduced pressure toyield benzhydryl7-amino-7-[(dimethylamino)methoxyphosphinyl]cephalosporanate.

Step C: Benzhydryl7-[(Dimethylamino)Methoxyphosphinyl]-7-α-(2-Furylacetamido)Cephalosporanate

A solution of benzhydryl7-[(dimethylamino)methoxyphosphinyl]cephalosporanate (280 mg.) inmethylene chloride (15 ml.) is stirred at 0° C. and 2-furylacetylchloride (145 mg.) is added. Pyridine (0.5 ml.) is then addedimmediately and the stirring at 0° C. continued for 15 minutes. Thereaction mixture is poured onto crushed ice which contains a slightexcess of hydrochloric acid. The layers are separated and the organicphase is washed with an excess of cold, dilute aqueous sodiumbicarbonate; it is then washed once more with water, dried andconcentrated. The crude product is purified by chromatography on silicagel with chloroform as the elution solvent. Concentration of the eluateyields benzhydryl7-[(dimethylamino)methoxyphosphinyl]-7-β-(2-furylacetamido)cephalosporanatetogether with a small amount of the isomer benzhydryl7-[(dimethylamino)methoxyphosphinyl]-7-α-(2-furylacetamido)cephalosporanate.

Step D: Sodium7-[(Dimethylamino)Methoxyphosphinyl]-7-β-(2-Furylacetamido)Cephalosporanate

A solution of benzhydryl7-[(dimethylamino)-methoxyphosphinyl]-7-β-(2-furylacetamido)cephalosporanate(65 mg.) and anisole (11 mg.) in methylene chloride (0.5 ml.) is stirredat 0° C. A cold solution of 23 mg. of trifluoroacetic acid (23 mg.) inmethylene chloride (0.2 ml.) is added over a period of 3-4 minutes.After an additional 30 minutes at 0° C. the mixture is concentrated todryness at greatly reduced pressure and the residue is taken up inbenzene (5 ml.) and 3 ml. of an aqueous solution of sodium bicarbonate(8.4 mg.) is added. The mixture is well stirred at room temperature forabout15 minutes and the layers are separated. The organic phase isextracted once with water and the combined aqueous phases are washedonce with ether and are lyophilized to yield sodium7-[(dimethylamino)methoxyphosphinyl]-7-β-(2-furylacetamido)cephalosporanate.

EXAMPLE 162 Disodium7-β-(α-Carboxyphenyl)Acetamido-7-(Bis(Dimethylamino)Phosphinyl]CephalosporanateStep A: Benzhydryl 7-Azido-7-[Bis(Dimethylamino)Phosphinyl]Cephalosporanate

A mixture of benzhydryl 7-Azido-7-bromocephalosporanate (545 mg.),tetramethyldiamidophosphorous acid (273 mg.) and silvertetrafluoroborate (195 mg.) in dimethoxyethane (5 ml.) is stirredovernight at room temperature protected from light, air, and moisture.The reaction mixture is filtered and the filtrate is concentrated todryness at reduced pressure. The residue is redissolved in methylenechloride and the solution is washed with a cold, dilute solution ofsodium bicarbonate and then dried and concentrated. The residue isdissolved in a little chloroform and charged onto a column of silica gelfrom which the product is eluted by chloroform. The eluate isconcentrated at reduced pressure to yield benzhydryl7-azido-7-[bis(dimethylamino)phosphinyl]cephalosporanate.

Step B: Benzhydryl7-amino-7-[bis(dimethylamino)phosphinyl]cephalosporanate

Benzhydryl 7-azido-7-[bis(dimethylamino)phosphinyl]cephalosporanate (250mg) is dissolved in dioxane (25 ml.) and hydrogenated at roomtemperature and normal pressure for 3/4 hour using platinum oxide (250mg) as a catalyst. A fresh portion of catalyst (250 mg) is added and thehydrogenation is continued for an additional 2-3 hours or until theazide has disappeared when examined in an infrared absorptionspectrophotometer. The mixture is concentrated to dryness at reducedpressure and the residue is taken up in chloroform and filtered bysuction through a layer of silica gel to remove the spent catalyst. Thesilica gel is then thoroughly washed with chloroform and the filtrate isconcentrated at low temperature and pressure to afford benzhydryl7-amino-7-[bis(dimethylamino)phosphinyl]cephalosporanate.

Step C: Benzhydryl 7-β-(α-tert.butoxy-carbonylphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]cephalosporanate

The benzhydryl 7-amino-7-[bis(dimethylamino)phosphinyl]cephalosporanateobtained in Step B is dissolved in methylene chloride (20 ml.) andα-(tert-butoxycarbonyl)phenylacetyl chloride (510 mg.) is added to thestirred solution at 0° C. The addition is immediately followed by theaddition of pyridine (1 ml.). After 15 minutes the reaction is quenchedby pouring onto crushed ice and the organic phase of the mixture iswashed successively with cold dilute hydrochloric acid, with cold dilutepotassium bicarbonate solution and then with water. The mixture is driedand concentrated at reduced pressure to yield benzhydryl7-β-(α-tert-butoxycarbonylphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]cephalosporanate.The product is purified by chromatography on silica gel, the purifiedmaterial being eluted with chloroform. The isomer benzhydryl7-α-(α-tert-butoxycarbonylphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]cephalosporanateis also obtained from this chromatographic purification.

Step D: Disodium7-β-(α-carboxyphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]-cephalosporanate

A mixture of benzhydryl7-β-(α-tert-butoxycarbonylphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]cephalosporanate(158 mg.), anisole (22 mg.) and methylene chloride (1 ml.) is stirred at0° C. A cold solution of 46 mg. of trifluoroacetic acid (46 mg.) inmethylene chloride (0.5 ml.) is added over a period of about 5 minutesand is concentrated to dryness at reduced pressure. The residue is takenup in a mixture of benzene (5 ml.) and ether (5 ml.) and 10 ml. of anaqueous solution of sodium bicarbonate (33.6 mg.) is added. Afterstirring at room temperature for 10 minutes the layers are separated andthe organic phase is again stirred with water (5 ml.) for an additional5 minutes. The combined aqueous extracts are washed once with ether (2ml.) and are lyophilized to yield disodium7-β-(α-carboxyphenyl)acetamido-7-[bis(dimethylamino)phosphinyl]cephalosporanate.

EXAMPLE 163 Trisodium7-Phosphono-7-β-(2-Thienylacetamido)Cephalosporanate Step A: Benzhydryl7-Azido-7-(Di-Tert-Butylphosphono)Cephalosporanate

A mixture of benzhydryl 7-azido-7-bromocephalosporanate (545 mg.),di-tert-butylphosphite (388 mg.) and silver tetrafluoroborate (195 mg.)in dimethoxyethane (5 ml.) is stirred overnight at room temperaturewhile protecting from air, light, and moisture. The mixture is thenfiltered from insoluble material and concentrated to dryness at lowtemperature and pressure. The residue is redissolved in methylenechloride and the solution is washed with a little ice water and thendried and concentrated. The residue is dissolved in a little chloroformand the solution is chromatographed on acid-washed alumina using amixture of chloroform and ethanol as the eluting solvent. The eluate isconcentrated at reduced pressure to afford benzhydryl7-azido-7-(di-tert-butylphosphono)cephalosporanate.

Step B: Benzhydryl 7-Amino-7-Di-tert-Butylphosphono)Cephalosporanate

Benzhydryl 7-azido-7-(di-tert-butylphosphono)cephalosporanate (110 mg.)is dissolved in dioxane (10 ml.) and is hydrogenated at room temperatureand under normal pressure for one hour using a platinum oxide catalyst(100 mg.). A second portion of catalyst (100 mg.) is added and thehydrogenation is continued for an additional 2-3 hours. The solvent iscompletely removed at reduced pressure and the residue in re-dissolvedin chloroform. The spent catalyst is then removed by filtration througha layer of silica gel and the filter bed is washed thoroughly withchloroform. The filtrate is concentrated at reduced pressure to yieldbenzhydryl 7-amino-7-(di-t-butylphosphono)cephalosporanate, which issuitable for use in the next step without further purification.

Step C: Benzhydryl7-Di-t-Butylphosphono-7-β-(2-Thienylacetamido)Cephalosporanate

The benzhydryl 7-amino-7-(di-t-butylphosphono)cephalosporanate obtainedin Step B is dissolved in methylene chloride (10 ml.) and the solutionis stirred at 0° C. To this solution is added 2-thienylacetic acid (32mg.) and dicyclohexylcarbodiimide (41 mg.). The reaction mixture is thenallowed to warm slowly to room temperature and is stirred overnight. Thedicyclohexylurea which forms is removed by filtration and the filtrateis concentrated at reduced pressure. The residue is dissolved in alittle chloroform and the solution is charged onto a column ofacid-washed alumina, from which benzhydryl7-di-tert-butylphosphono-7-β-(2-thienylacetamido)cephalosporanate isobtained by elution with a dilute solution of methanol in chloroform.The product is recovered by concentration of the eluate. A small amountof the isomer benzhydryl7-di-tert-butylphosphono-7-α-(2-thienylacetamido)cephalosporanate isalso obtained.

Step D: Trisodium 7-Phosphono-7-(α-2-Thienylacetamido)Cephalosporanate

A mixture of benzhydryl7-di-tert-butylphosphono-7-β-(2-thienylacetamido)cephalosporanate (70mg.), anisole (33 mg.) and methylene chloride (1 ml.) is stirred at 0°C. and a cold solution of trifluoroacetic acid (70 mg.) in methylenechloride (0.2 ml.) is added. After about 30 minutes the solution isconcentrated to dryness at reduced pressure and the residue is suspendedin a mixture of benzene (3 ml.) and ether (3 ml.) and 5 ml. of anaqueous solution of sodium bicarbonate (50 mg.) is added. The mixture isstirred well and the layers are separated. The organic phase is washedwith 5 ml. of water and the combined aqueous solutions are washed withether and lyophilized to yield trisodium7-phosphono-7-β-(2-thienylacetamido)cephalosporanate.

EXAMPLE 164

By following the procedures described in Example 163, Steps A-D all ofthe 7-amido-7-phosphonocephalosporanic acid salts of this invention maybe obtained. Thus, for example, by substituting an equivalent amount ofthe appropriate phosphite and acyl halide for thedi-tertiarybutylphosphite and 2-thienylacetic acid reactants recited inSteps A and C of Example 163 and otherwise following the proceduredescribed therein, the corresponding salts of7-amido-7-phosphonocephalosporanic acid are obtained. The followingequation and accompanying Table IV illustrate this method ofpreparation: ##STR92##

                  TABLE IV                                                        ______________________________________                                          R             R.sup.5   X      M    Base                                    ______________________________________                                                      C.sub.2 H.sub.5                                                                           Br     Na   NaOH                                    HOOCCH.sub.2  (CH.sub.2).sub.2 CH.sub.3                                                                 Cl     K    KHCO.sub.3                               ##STR93##    CH.sub.3    Br     Na   NaHCO.sub.3                             HOOCCH(C.sub.6 H.sub.5)                                                                     C.sub.2 H.sub.5                                                                           Cl     Li   LiOH                                    HOOCCH(C.sub.6 H.sub.5)                                                                     (CH.sub.2).sub.3 CH.sub.3                                                                 I      Na   Na.sub.2 CO.sub.3                       ______________________________________                                    

EXAMPLE 165 Sodium 7-(β-cyanoethyl)-7-(2-furylacetamido)cephalosporanate

A solution of 500 mg. of benzhydryl7-(p-nitrobenzylideneamino)cephalosporanate prepared as in Example 43 ina mixture of 2.5 ml. of t-butyl alcohol and 2.5 ml. of acrylonitrile istreated with 20 μl. of N,N-diisopropylethylamine with stirring undernitrogen. The initially green solution turns yellow-orange afterstirring 10 minutes. It is then concentrated under reduced pressure to620 mg. of a yellow gummy residue. This residue is chromatographed on amixture of 48 g. of active, powdered silica gel and 40 g. of powdereddiatomaceous earth. Elution is carried out with 21 ether in benzene. Thedesired non-crystalline cyanoethyl adduct is eluted after 2 liters ofeluent has passed through the column. The product is identified as7-(p-nitrobenzylideneamino)-7-(β-cyanoethyl)cephalosporanic acidbenzhydryl ester.

Using the procedures described in the foregoing examples, the aboveproduct is converted to sodium7-(β-cyanoethyl)-7-(2-furylacetamido)cephalosporanate.

EXAMPLE 166 Sodium 7-methyl-7-(2-thienylacetamido)cephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate (571 mg.) isdissolved in 10 ml. dry acetonitrile containing 0.950 ml. dimethylsulfate. With vigorous stirring under nitrogen, a solution ofdiisopropylethylamine in 12 ml. acetonitrile is added over a 5 hourperiod. The reaction mixture is stirred overnight and the solventremoved in vacuo. The residue is taken up in 50 ml. benzene and washedsuccessively with water, 1 M aqueous pH 2 phosphate buffer, water, andbicarbonate. After drying, filtering and removing the solvent, theresidue is a crude compound suitable for further reaction withoutadditional purification. It may be purified if desired by chromatographyon silica gel, eluting with 25:1 chloroform-ethylacetate. It isidentified as 7-(p-nitrobenzylideneamino)-7-methylcephalosporanic acidbenzhydryl ester.

This product is converted to the title compound following proceduresdescribed in the foregoing examples.

EXAMPLE 167 Sodium 7-acetyl-7-(2-thienylacetamido)cephalosporanate

Benzhydryl 7-(p-nitrobenzylideneamino)cephalosporanate (571 mg.) isdissolved in 10 ml. dry acetonitrile containing 0.950 ml. acetylchloride. With vigorous stirring under nitrogen, a solution ofdiisopropylethylamine in 12 ml. acetonitrile is added over a 5 hourperiod. The reaction mixture is stirred overnight and the solventremoved in vacuo. The residue is taken up in 50 ml. benzene and washedsuccessively with water, 1 M aqueous pH 2 phosphate buffer, water andbicarbonate. After drying, filtering and removing the solvent, theresidue is a crude compound suitable for further reaction withoutadditional purification. It may be purified if desired by chromatographyon silica gel eluting with 25:1 chloroform-ethylacetate. It isidentified as 7-(p-nitrobenzylideneamino)-7-acetylcephalosporanic acidbenzhydryl ester.

This product is converted to the title compound following proceduresdescribed in the foregoing examples.

EXAMPLE 1683-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid Step A:7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

The mono-sodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (20.5 gm.) is dissolved in the mixture of acetone (80 ml.) andaqueous 10% dipotassium hydrogen phosphate (240 ml.). To this solutionis added dropwise trichloroethoxycarbonyl chloride (25 gm., 118 mmoles)in acetone (80 ml.). During the addition the pH of the solution is keptat 9.1 by gradual addition of 2.5 N sodium hydroxide solution. After 30minutes the mixture is extracted with ethyl acetate, the ethyl acetatelayer discarded, and the aqueous layer is acidified to pH 2.5 withconcentrated hydrochloric acid. The precipitated product is extractedinto ethyl acetate. After drying over sodium sulfate and removing thesolvent in vacuo, the title compound is obtained as an oil.

UV: (CH₃ OH) λmax. 262.5 ε=5450

NMR: (Solvent--DMSO, d₆) δ=3.43 (O--CH₃, s), 4.73 (2-H₂, partiallyvisible), ##STR94## 5.12 (6-H, s), ˜4.74 (10-H₂, partially visible).

Step B: Di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

To the solution of the above7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in ethyl acetate (500 ml.) is added diphenyldiazomethane (17 gm.)in 200 ml. of ether. After agitating the mixture overnight, it isextracted successively with sodium bicarbonate and sodium chloridesolutions. The solvent is evaporated from the dried solution to afford acrude product which is purified by chromatography on silica gel. A 2:1mixture of chloroform and ethyl acetate is used for elution. Thismaterial showed a single spot on TLC chromatography.

UV: (CH₃ OH) λmax. 2650 μm ε7000

NMR: (Solvent--CDCl₃) δ=3.45 (O--CH₃, s), 3.35 (2-H₂, partially visible,##STR95## 5.03 (6-H, s), ˜4.88 (10-H₂, partially visible).

Step C: Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (1.1 gm., 1.18 mmole), acetonitrile (5 ml.) and bis-trimethylsilyltrifluoroacetamide (3 ml.) is allowed to stand at room temperature for 6hours. After this period, the volatile products are removed in highvacuum and the residue is dissolved in 3 ml. of methylene chloride. Tothis solution is added phenylacetyl chloride (0.23 ml., 1.79 mmole) andthe mixture is allowed to stand at room temperature for 65 hours. Afterthis, the solution is evaporated and the residue is dissolved in 5 ml.tetrahydrofuran and 0.7 ml. of 2.5 N hydrochloric acid. After 20 minutesreaction time the solvent is evaporated and the residue is partitionedbetween methylene chloride and sodium bicarbonate solution. The organiclayer is washed with sodium chloride solution, dried and evaporated todryness. The crude product thus obtained is purified by chromatographyon silica gel, using chloroform ethyl acetate 95:5 as eluant. The titlecompound obtained appears homogenous on thin layer chromatography.

UV: (CH₃ OH) λmax. 2640 μm ε6650

NMR: (Solvent--CDCl₃) δ=3.50 (O--CH₃, s), 3.31 (2-H₂, partiallyvisible), ##STR96## 5.04 (6-H, s), ˜4.96 (10-H₂, partially visible),3.95 (13-H₂, s).

Step D: Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

The solution of di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (104 mg.) in 90% acetic acid-water (1 ml.) is agitated with 100 mg.of zinc dust for 5 hours. After this, the solution is filtered and thesolvent is removed in vacuo. The residue is partitioned betweenmethylene chloride and water, and the methylene chloride layer isextracted with sodium bicarbonate and sodium chloride solutions. Afterdrying and evaporation a crude product is obtained which is purified bythin layer chromatography utilizing silica gel plates and a 3:2 mixtureof chloroform and ethyl acetate. The product is characterized by its IRand NMR spectra.

IR: (CHCl₃) 1780, 1730 and 1680 cm⁻¹

UV: (CH₃ OH) λmax. 2640 μm ε5870

NMR: (Solvent--CDCl₃) δ=3.40 (O--CH₃, s), 3.33 (2-H₂, partiallyvisible), 5.01 (6-H, s), ˜4.88 (10-H₂, partially visible), 3.60 (13-H₂,s).

Step E:3-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicacid (17 mg.) is dissolved in anisole (0.2 ml.) and treated withtrifluoroacetic acid (0.5 ml.) for 5 minutes. After this period, themixture is concentrated rapidly in high vacuum and diluted with ethylacetate. The product is removed from the ethyl acetate solution byextraction with a pH 7.5 sodium phosphate buffer. The buffer solution isacidified to pH 2.5 with dilute hydrochloric acid and the title compoundis removed by extraction with ethyl acetate. After drying andevaporating the solution, the product is obtained. An analytical sampleis obtained by recrystallization from ethylacetate. MP: 159°-161° C.

UV: (pH 7 buffer) λmax. 2670 μm ε8650

IR: (CH₃ CN) 1780, 1735 and 1700

NMR: (Solvent--CD₃ CN+D₂ O) δ=3.42 (O-CH₃, s), 3.35 (2-H₂, partiallyvisible), 5.01 (6-H, s), 4.83 (10-H₂, d), 3.61 (13-H₂, s).

Elemental analysis for C₁₈ H₁₉ O₇ N₃ S: Calc: C, 51.29; H, 4.54; Found:C, 51.47; H, 4.73.

Two milligrams of the above acid is dissolved in one drop of methanoland treated with a solution of 2 mg. dibenzyl ethylenediamine diacetatein ethyl acetate. The dibenzyl ethylenediamine salt of the titlecompound precipitates after standing in the form of needle-likecrystals. MP: 140°-143° C.

UV: (CH₃ OH) λmax. 263 μm ε8600

Preparation of Monosodium Salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylicAcid Modified Fermentation Process Step 1: Slants

A lyophilized tube of Streptomyces lactamdurans culture (MA-2908) wasopened asceptically and the organism transferred to a medium of thefollowing composition:

Medium XI

1% Blackstrap Molasses

1% National Brewer's Yeast

2.5% Difco agar pH 7.0

Water to volume

The slants are inoculated for seven days at 28° C. When stored in thecold, the slants are stable for more than 13 weeks.

Step 2: Seed Stages: Two Stage System

First Seed: The first seed is inoculated directly from the slant of Step1 to 40 ml. of 1% Primary Dried Yeast N.F., pH 7.0 (obtained from theYeast Product Corporation) in a 250 ml. baffled Erlenmeyer flask. Theflasks were then shaken on a 220 rpm. rotary shaker with a 2 inch throwat 28° C. for a period of from two to three days.

Second Seed: A 2.5% inoculum from the first seed stage was added to aflask containing a 2% Fleischmann S-150 yeast autolysate, pH 7.0. Thegrowth in this stage is characteristically light and the incubation,performed as in the first stage, was not extended beyond 48 hours.

Step 3: Production Medium

The production medium contains per liter of distilled water: 30 g.distiller's solubles; 7.5 g. of Primary Dried Yeast N.F. and 0.25% v/vMobilpar-S defoamer. The medium is adjusted to pH 7.0 with a smallamount of concentrated sodium hydroxide solution, dispensed intoErlenmeyer flasks and autoclaved for 15 or 20 minutes at 121° C. Aftercooling the medium received a 2.5% inoculum of the seed obtained in Step2. The time of incubation can vary from about 50 hours to 100 hours butan incubation period of about 72 hours is preferred. The volume of mediain each flask can vary from 30 to 50 ml. but 40 ml. was used routinely.The level of inoculum can vary from 1% to 5%; but, in practice, a 2.5%level is generally employed.

Step 4: Assay

When the fermentation was complete, the cells were removed bycentrifugation and the broth was diluted with phosphate buffer, pH 7.0.The concentration of7β-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylicacid in the fermentation broth was determined by the standardbiological-disc assay method. The assay organism employed was Vibriopercolans (ATCC 8461). Filter paper discs are emersed into the dilutedbroths and placed on the surface of agar-containing Petri dishes thathad been inoculated with the assay organism Vibrio pecolans (ATCC 8461).Also placed on these Petri dishes are discs that had been dippedpreviously in standard solutions containing known concentrations of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylicacid. The discs were incubated overnight at 28° C. and the diameters ofthe zones of inhibition recorded. The concentration of product and thefermented broth is calculated by interpolation from the standard curvewhich relates zone diameter with the known concentrations of standardsolutions of the product. By this procedure it was calculated thatStreptomyces lactamdurans MB-2908 produced 78.6 μg./ml. of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in the modified fermentation process.

Step 5: Isolation

The filtered broth is adjusted to pH 7.0 with dilute hydrochloric acidand 2900 ml. is passed through a column containing a strongly basicanion exchange resin (100 g.) having a styrene-divinylbenzene matrix(Dowex 1×2 chloride cycle resin) at 10 ml./minute. The spent solvent iscollected in 500 ml. fractions. The resin column is washed with waterand eluted with 3% ammonium chloride in 90% methanol. The eluate iscollected in 100 ml. fractions. The spent fractions are combined, the pHadjusted to pH 7.2 to 8.0 with dilute sodium hydroxide and adsorbed on astrongly basic anion exchange resin (100 g.) having astyrene-divinylbenzene matrix (Dowex 1×2 chloride cycle resin) at 14ml./minute. The column is washed with water and eluted with 5% aqueoussodium chloride. The eluate is collected in 50 ml. fractions andconcentrated. The concentrate is diluted to 500 ml., adjusted from pH8.8 to pH 2.0 with dilute hydrochloric acid and adsorbed on 25 ml. of astrongly acidic cation exchange resin of the sulfonate type having astyrenedivinylbenzene matrix (Dowex 50×2 hydrogen cycle resin) at 2.5ml./minute. The column is washed with 25 ml. of water then eluted with2% pyridine until the pH of the column effluent rose to pH 7 (54 ml.).The eluate thus obtained is adjusted to pH 8.0 with dilute sodiumhydroxide and concentrated under vacuum to remove the pyridine andafford the monosodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid.

Elemental analysis for C₁₆ H₂₁ N₄ SO₉ Na: Calc.: C, 41.0%; H, 4.5%; N,12.0%; S, 6.8%; Found: C, 39.31%; H, 4.76%; N, 11.16%; S, 6.46%.

EXAMPLE 1693-Carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid Step A: Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A solution of the di-benzhydryl ester of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (9.3 gm., 10 mmoles), N-trimethylsilyl phthalimide (7.8 gm., 40mmoles) and phenylacetyl chloride (5.3 ml., 40 mmoles) in 50 ml. ofacetonitrile is heated to 40° C. for 20 hours. After this period themixture is cooled to room temperature and filtered. The filtrate isevaporated to dryness and triturated with hexane. The insoluble residue,containing di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)phenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, is used without purification in the next step.

Step B: Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-phenylacetamido-3-cephem-4-carboxylicAcid

The crude product from Step A is dissolved in a mixture of ethylacetate(50 ml.), acetic acid (45 ml.) and water (5 ml.). To this solution isadded 20 gm. of zinc powder and the mixture is agitated at roomtemperature for 4 hours. After this, the excess zinc is removed byfiltration and the filtrate partitioned between ethylacetate and water.The organic layer is washed with a sodium bicarbonate solution andwater, dried and the solvent is evaporated. The crude product thusobtained is purified by chromatography on 1 kg. of silica gel, using amixture of chloroform, hexane, and methanol (47:47:6) for elution. Theproduct obtained has the physical characteristics described in Example168, Step E.

Step C:3-Carbamoyloxymethyl-7-methoxy-7-phenylacetamido-3-cephem-4-carboxylicAcid

The title compound is prepared by the procedure described in Example 168Step F, and has the same physical characteristics as the product ofExample 168.

EXAMPLE 1703-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid Step A: Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-thienylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of 6.0 gm. (6.3 mmole) of the dibenzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid, 4.7 gm. (40 mmoles) N-trimethylsilyl trifluoroacetamide, 3.42 ml.(25 mmoles) 2-thienylacetylchloride, and 50 ml. of chloroform is warmedat 47° C. for 16 hours. After the solvent is removed by evaporation, thecrude reaction mixture is extracted with hexane, and further purified bychromatography on 1 kg. of silica gel using 10% ethylacetate inchloroform as the eluant.

UV: (CH₃ OH) λmax. 265 μm ε5810

NMR: (Solvent--CDCl₃) δ=3.53 (--OCH₃, s), ˜3.4 (2-H₂, d), ##STR97## 5.05(6-H, s), ˜5.0 (10-H₂, partially visible), 4.15 (13-H₂, s).

Step B: Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

The di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-3-thienylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (4.2 gm., 3.8 mmoles) is dissolved in 30 ml. of ethylacetate andadded to 30 ml. of 90% aqueous acetic acid and 12 gm. of zinc dust. Themixture is stirred vigorously for 51/2 hours at room temperature. Afterthe zinc is filtered off, excess acetic acid is removed by washing theethylacetate solution with water. The title compound is isolated in thesame manner as described in Example 1, Step E. It is characterized byTLC (7% CH₃ OH in 1:1 CHCl₃ :n-hexane) as a single spot material.

IR: (CHCl₃) 1740, 1800 cm⁻¹ ;

UV: λmax. 263 μm ε5800

NMR: (Solvent--CDCl₃) δ=3.45 (--OCH₃, s), ˜3.4 (2-H₂, d), 5.02 (6-H, s),˜4.92 (10-H₂, partially visible), 3.85 (13-H₂, s).

Step C:3-Carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

A cold solution of the benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamido)-3-cephem-4-carboxylicacid (1.36 gm.) in 10.88 ml. of anisole is stirred with 5.44 ml. oftrifluoroacetic acid at 0° C. for 1/2 hour. The volatiles are removed inhigh vacuum, and the product is recrystallized from ethyl acetate. MP:165°-167° C.

    __________________________________________________________________________    UV: (pH 7 buffer)                                                                      λmax. 263 μm                                                                ε8840                                                                      ;α].sub.D (C = 1, CH.sub.3 OH)                                          = + 199°                                                  236μm                                                                              ε14000                                               __________________________________________________________________________

NMR: (Solvent--CD₃ CN+D₂ O) δ=3.48 (--OCH₃, s), ˜3.4 (2-H₂, partiallyvisible), 5.05 (6-H, s), 4.91 (10-H₂, d), 3.86 (13-H₂, s).

Elemental analysis for C₁₆ H₁₇ N₃ O₇ S₂ : Calc.: C, 44.96; H, 4.01; N,9.83 Found: C, 44.86; H, 3.99; N, 9.21, S, 15.00.

Step D: Sodium3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamide)-3-cephem-4-carboxylate

A suspension of 1 gram of3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamide)-3-cephem-4-carboxylicacid in 100 ml. of distilled water is stirred at room temperature whilegradually adding 0.2 gram of sodium bicarbonate. After solution isattained and pH is substantially neutral (pH 6-7), the batch is filteredinto a lyophilization bottle. The filtrate is lyophilized.

There is obtained 1 gram of amorphous sodium3-carbamoyloxymethyl-7-methoxy-7β-(2-thienylacetamide)-3-cephem-4-carboxylate,representing a recovery of 99%. UV (pH 7 buffer): E% 198 at 262 nm, 315at 236 nm. ir (KBr): 1760 (lactam). [α]_(D) =183.1° (C=1, pH 7 buffer).

EXAMPLE 1713-Carbamoyloxymethyl-7β-(2-furylacetamido)-7-methoxy-3-cephem-4-carboxylicacid

Step A: Di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-furylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (9.3 gm.), bis-(trimethylsilyl)-trifluoroacetamide (7.0 ml.),2-furylacetylchloride (4.7 ml.) and dichloromethane (50 ml.) is warmedat 47° C. for 16 hours. The solvent is removed by evaporation, the crudereaction mixture is extracted with hexane, and the residue is usedwithout purification in the next step.

NMR: (Solvent-CDCl₃) δ=3.48 (--OCH₃, s), 3.08 (2-H₂, d), ##STR98## 5.02(6-H, s), ˜4.88 (10-H₂, d), 3.72 (13-H₂, s).

Step B: Benzhydryl ester of7β-(2-furylacetamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

The di-benzhydryl ester from Step A is reacted with zinc dust and aceticacid following the procedures described in Example 170 Step B. Aftercrystallization from chloroformhexane, the pure product has thefollowing physical characteristics:

MP: 168°-171° C.

IR: (CHCl₃) 1800, 1720, 1700

UV: λmax. 265 μm ε7200

NMR: (Solvent --CD₃ CN) δ=3.43 (--OCH₃, s), 3.39 (2-H₂, partiallyvisible), 5.0 (6-H, s), 4.75 (10-H₂, d), 3.64 (13-H₂, s).

Step C:3-Carbamoyloxymethyl-7-methoxy-7β-(2-furylacetamido)-3-cephem-4-carboxylicAcid

The3-carbamoyloxymethyl-7-methoxy-7β-(2-furylacetamido)-3-cephem-4-carboxylicacid is prepared from the product of Step B following the proceduredescribed in Example 170 Step C. The product, after recrystallizationfrom ethyl acetate, has a melting point of 156°-161° C.

UV: (pH 7 buffer) λmax. 265 μm ε7200

IR is consistent with the structure.

NMR: (Solvent-CD₃ CN+D₂ O) δ=3.44 (--OCH₃, s), ˜3.38 (2-H₂, partiallyvisible), 5.02 (6-H, s), 4.82 (10-H₂, d), 3.66 (13-H₂, s).

EXAMPLE 1723-Carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid Step A: Di-benzhydryl ester of7β-[(D-5-Trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

By following substantially the procedure described in Example 171 StepA, and by substituting for the 2-furylacetyl chloride an equimolarquantity of phenylthioacetyl chloride there is obtained di-benzhydrylester of7β-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid.

NMR: (Solvent-CDCl₃) δ=3.33 (--OCH₃, s), ˜3.23 (2-H₂, partiallyvisible), ##STR99## 5.0 (6-H, s), 4.87 (10-H₂, u), 3.68 (13-H₂, s).

Step B: Benzhydryl ester of3-carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid

By following substantially the procedure described in Example 171 StepB, and by substituting the di-benzhydryl ester of7β-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)thiophenoxyamido]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in place of the di-benzhydryl ester of7β-[(D-5'-trichloroethoxycarbonylamino-5'-carboxyvaleryl)-2-furylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid there is obtained, after chromatographic purification,substantially pure product which appears as a single spot on thin layerchromatography (TLC). The IR is in accord with the structure.

UV: λmax. 274 μm ε11350

NMR: (Solvent-CDCl₃) δ=3.34 (--OCH₃, s), 3.24 (2-H₂, partially visible),5.0 (6-H, s), 4.88 (10-H₂, d), 3.68 (13-H₂, s).

Step C:3-Carbamoyloxymethyl-7-methoxy-7β-thiophenoxyacetamido-3-cephem-4-carboxylicAcid

The title compoun is prepared from the product of Step B above followingthe procedure of Example 170 Step C. The product exhibits a single spoton TLC. MP: 119°-123° C.

UV: (pH 7 buffer) λmax. 247 μm ε10400

NMR: (Solvent-CD₃ CN+D₂ O) δ=3.38 (--OCH₃, s), 3.34 (2-H₂, partiallyvisible), 5.0 (6-H, s), 4.82 (10-H₂, s), 3.71 (13-H₂, s).

EXAMPLE 1737β-(D,L-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid Step A:7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (50.0 g.) is dissolved in a mixture of 1500 ml. aqueous 5%dipotassium hydrogen phosphate and 1000 ml. acetone and adjusted to pH9.5 with 2.5 N sodium hydroxide solution. To this stirred solution isadded tert-butoxycarbonyl azide (50 ml.) and the pH maintained at 9.5over a 20 hour period. The reaction mixture is then extracted with ethylacetate, the ethyl acetate layer discarded, and the aqueous layer iscooled to 0° C., stirred with 1200 ml. of ethylacetate, and acidified topH 2.5 with concentrated hydrochloric acid. The ethyl acetate layer isseparated, dried over sodium sulfate and concentrated in vacuo, and thesolid so obtained may be used without further purification.

IR: 1790 (β-Lactam), 1700

UV: (pH 7 buffer) λmax. 263 ε6820

NMR: (Solvent-DMSO, d₆) δ=3.30 (--OCH₃, s), 3.42 (2-H₂, partiallyvisible), 5.06 (6-H, s), 4.78 (10-H, d), 1.38 (t-Bu, s).

Step B: Di-benzhydryl ester of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-butoxycarbonylamino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (15.0 g.) in ethyl acetate (500 ml.) is added diphenyldiazomethane(5.5 g.) in 70 ml. of ether. The reaction mixture is warmed to 40° C.with stirring and after 30 minutes is treated with additionaldiphenyldiazomethane (5.5 g.) in ether (70 ml.). After 3 hours, thesolvent is removed in vacuo and replaced by a mixture of methanol (500ml.) and water (20 ml.). The methanol-water solution is extracted fourtimes with hexane and then evaporated in vacuo. The residue is dissolvedin ethyl acetate, dried over sodium sulfate and evaporated in vacuo toyield the title compound which is used without purification in the nextstep.

NMR: (Solvent-CDCl₃) δ=3.60 (--OCH₃, s), 3.4 (2-H₂, partially visible),5.10 (6-H, s), 4.95 (10-H, partially visible).

Step C: Di-benzhydryl ester of7β-[(D-5'-tert-butoxycarbonylamino-5'-carboxyvaleryl)-D,L-α-azidophenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A mixture of the di-benzhydryl ester of7β-(D-5'-tert-butoxycarbonylamino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (10.8 g.), chloroform (100 ml.),bis-(trimethylsilyl)-trifluoroacetamide (16.2 g.) andD,L-α-azido-phenylacetyl chloride is warmed at 45° C. for 16 hours. Themixture is diluted with chloroform (300 ml.), washed with 2% aqueousbicarbonate and saturated aqueous sodium chloride, dried over sodiumsulfate and evaporated to an oil which is purified by precipitating theproduct from a chloroform solution with hexane. The light yellow solidis used in the next step without further purification.

IR: 1790 (β-Lactam, 1, 1735, 2100 (--N₃)

NMR: (Solvent-CDCl₃) δ=3.70 (--OCH₃, s), 3.2 (2-H₂, partially visible).

Step D:7β-(D,L-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A solution of the di-benzhydryl ester of7β-[(D-5'-tert-butoxycarbonylamino-5'-carboxyvaleryl)-D,L-α-azidophenylacetylamino]-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (13.0 g.) in anisole (13 ml.) is poured into 65 ml. of cold (0° C.)trifluoroacetic acid. After 5 minutes the solution is poured into 1800ml. of stirred, cold (0° C.) ether. The precipitated solid is collectedand distributed between 10% aqueous disodium acid phosphate and ethylacetate. The ethyl acetate layer is discarded and the aqueous layer islayered with fresh ethyl acetate and the stirred mixture brought to pH 2in the cold with 60% aqueous phosphoric acid. The ethyl acetate layer iscollected, washed with saturated aqueous sodium chloride and then driedover sodium sulfate. Volatiles are removed in vacuo to afford the titlecompound.

    ______________________________________                                        UV: λmax. 264μm                                                                     ε7537                                                                            (pH 7 buffer)                                      231 μm       ε13567                                                ______________________________________                                    

IR: 1760 (β-Lactam) 1705, 2105 (--N₃)

NMR: (Solvent-CD₃ CN) δ=3.36 (--OCH₃, s), 3.50 (--OCH₃, s), 3.40 (2-H₂,partially visible), 5.06 (6-H, s), 4.86 (10-H, s), 5.15 (13-H, s).

EXAMPLE 1747β-(D,L-α-aminophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicAcid

A slurry of 1.0 g. of7β-(D,L-α-azidophenylacetylamido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid in acetic acid (10 ml.) and water (90 ml.) at 0° C. is stirred withzinc dust (5.0 g.) for 10 minutes and filtered. The filtrate is spargedwith hydrogen sulfide, filtered, and the filtrate freeze dried to afforda white solid which is washed with ether and dried in vacuo to affordthe title compound as a white powder.

UV: (pH 7 buffer) λmax. 264 μm ε6525

IR: 1770 (β-Lactam) 2650, 1550 (HN₃ +)

NMR: (Solvent-D₂ O+HCO₃ -) δ=3.78 (--OCH₃, s), 3.84 (--OCH₃, s), 3.90(2-H₂, partially visible).

EXAMPLE 1753-Acetoxymethyl-7β-(2-thionylacetamido)-3-cephem-4-carboxylic Acid StepA:7β-(D-5-Trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-amino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicacid (2.5 g., 0.53 mole) in acetone (13 ml.) and aqueous 10% dipotassiumhydrogen phosphate (40 ml.) is added dropwise trichloroethoxycarbonylchloride (3.35 g., 0.159 mole). During the addition the pH of thesolution is kept in the range of from 8.5 to 9 by the gradual additionof a 17% aqueous solution of sodium hydroxide. After 30 minutes themixture is washed with ethyl acetate and the aqueous layer is acidifiedto pH 2.5 with concentrated hydrochloric acid. The precipitated productis extracted into ethyl acetate, the solution is dried over sodiumsulfate, filtered and the solvent removed to afford 2.7 g. of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid.

Step B: Dibenzhydryl ester of7-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicAcid

To a solution of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid in ethyl acetate (30 ml.) is added diphenyl diazomethane (2.0 g.)in ether (25 ml.). The mixture is stirred overnight and the solventremoved to afford 4.0 g. of crude product. The crude product is purifiedby chromatography on silica gel using chloroform as the eluant to afford2.3 g. of substantially pure dibenzhydryl ester of7-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetylmethyl-3-cephem-4-carboxylicacid.

NMR: (Solvent-CDCl₃) δ=2.0 (methyl, s), 4.9 (10-H₂, quartet), 3.2 (2-H₂,quartet), 4.95 (6-H, d), 5.92 (7-H), 7.0 (benzhydryl protons, 2 s).

Step C: Dibenzhydryl ester of7-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicAcid

A mixture of the dibenzhydryl ester of7β-(D-5-trichloroethoxycarbonylamino-5-carboxyvaleramido)-3-acetoxymethyl-3-cephem-4-carboxylicacid (2.0 g., 0.02 mole), N-trimethylsilyl trifluoroacetamide (1.65 g.,0.09 mole), 2-thienylacetyl chloride (1.31 g., 0.0815 mole) andmethylene chloride (6 ml.) is warmed at 40°-45° C. in an oil bath undera nitrogen atmosphere for 20 hours. The reaction mixture is poured intohexane (100 ml.) and filtered through diatomaceous earth. Removal of thesolvent affords the dibenzhydryl ester of7-[D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicacid.

Step D: Benzhydryl ester of3-acetoxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic Acid

The dibenzhydryl ester of7-[(D-5-trichloroethoxycarbonylamino-5-carboxyvaleryl)-2-thienylacetylamino]-3-acetoxymethyl-3-cephem-4-carboxylicacid is dissolved in ethyl acetate (10 ml.) and added to a mixture of90% aqueous acetic acid (10 ml.) and zinc dust (1.0 g.). The mixture isstirred for two hours at room temperature. The reaction mixture isfiltered to remove the zinc. The reaction mixture is washed successivelywith 2 portions of water, a cold sodium bicarbonate solution and thenwith a saturated sodium chloride solution (15.0 ml.). The ethyl acetatesolution is dried over sodium sulfate, filtered and the solvent removedto afford 1.9 g. of crude product which is chromatographed on silica gelusing a mixture of chloroform and ethyl acetate (50:1) as the eluant toafford 0.380 g. of product which, after recrystallization from ethylacetate, has a melting point of 141.5°-143° C.

UV: (CH₃ OH) λmax. 263 δ7580

Elemental analysis for C₂₉ H₂₆ N₂ O₆ S₂ : Calc.: C, 61.91; H, 4.66; N,4.98; Found: C, 62.14; H, 4.84; N, 4.91.

Step E: 3-(Acetoxymethyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylicAcid

A cold solution of benzhydryl ester of3-acetoxymethyl-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid (100mg.) in anisole (1.0 ml.) and trifluoroacetic acid (0.5 ml.) is stirredat 0° C. for 35 minutes. Carbon tetrachloride (50 ml.) is added and thereaction mixture is concentrated to dryness. The residue is trituratedwith hexane. The hexane is removed by decantation and this residue isdissolved in ethyl acetate (10 ml.), concentrated to 1 ml. and diethylether added to afford precipitate. This precipitate is recrystallizedfrom a mixture of diethyl ether and ethyl acetate to afford 0.025 g. of3-(acetoxymethyl)-7-(2-thienylacetamido)-3-cephem-4-carboxylic acid,m.p. 164° C. Mixed melting point with an authentic sample was 163° C.

EXAMPLE 176 Dibenzylethylenediamine salt of3-methyl-7-methoxy-7β-(2-thienylacetamido-3-cephem-4-carboxylic AcidStep A:7β-(D-5-Amino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicAcid

A 10% palladium on charcoal catalyst is suspended in water (80 ml.) andtreated with hydrogen. The catalyst is filtered and suspended again inwater (50 ml.) and to this mixture (2.67 g.) is added the sodium salt of7β-(D-5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid (1.0 g.) in water (10 ml.). The resulting mixture is shaken fortwenty-two hours at room temperature. The catalyst is removed byfiltration and washed with water (50 ml.). The combined wash andfiltrate is concentrated to dryness to afford a 52.8% yield of7β-(D-5-amino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicacid (528 mg.).

UV: λmax. 265 μm; E₁ cm^(1%) is 100

Step B: Dibenzylethylenediamine salt of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicAcid

A solution of the disodium salt of7β-(D-5-amino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid (11.5 g.) is dissolved in water (150 ml.) and acetone (50 ml.). ThepH is adjusted to 9-9.1 with sodium hydroxide and 10 ml. of tert-butylazidoformate is added. The reaction mixture is stirred for 16 hours atroom temperature with additional sodium hydroxide being added tomaintain the pH at 9-9.1. The reaction mixture is extracted with ethylacetate (100 ml.) and the organic layer discarded. The product isprecipitated by lowering the pH to 2.5 with dilute hydrochloric acid.The precipitate is collected by centrifugation and converted to itsdibenzylethylenediamine salt which is crystallized from ethyl acetate.There is obtained 4.3 g. of the dibenzylethylenediamine salt of7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid, m.p. 177°-179° C. (dec.).

UV: λmax. 263 μm, 238 E₁ cm^(1%) =98.2, 81.1

Elemental analysis for C₃₆ H₄₉ N₅ O₉ S: Calc.: C, 59.42; H, 6.74; N,9.63; Found: C, 60.02; H, 6.80; N, 9.79.

Step C: Dibenzylethylenediamine salt of3-methyl-7-methoxy-7β-(2-thienylacetamido-3-cephem-4-carboxylic Acid

The7β-(D-5-tert-butoxycarbonylamino-5-carboxyvaleramido)-3-methyl-7-methoxy-3-cephem-4-carboxylicacid is treated with aqueous dilute hydrochloric acid (200 ml., 0.1 N)and ethyl acetate (100 ml.) in order to extract the free acid. To asolution of 1.33 g. (2.74 mmoles) of the free acid in methylene chloride(10 ml.) is added bis-trimethylsilyl trifluoroacetamide (2.2 ml.) andmono-trimethylsilyl trifluoroacetamide (0.5 ml.). 2-Thienylacetylchloride (1.1 ml.) is then added and the reaction mixture stirred for 18hours under a nitrogen atmosphere at 43° C. The solvent is removed invacuo, and the residue partitioned between ethyl acetate and aqueousphosphate buffer (pH 7.5). The aqueous layer is acidified with dilutehydrochloric acid and the precipitated product extracted with ethylacetate. Addition of dibenzethylenediamine results in crystallization of250 mg. of the desired product as a salt in the proportion of 2equivalents of product to one mole of dibenzylethylenediamine.Recrystallization of the salt from ethanol affords substantially pureproduct, m.p. 153°-155° C. (dec.) with previous darkening.

Elemental analysis for C₄₆ H₅₂ S₄ N₆ O₁₀ : Calc.: C, 56.54; H, 5.36; N,8.60; S, 13.12; Found: C, 55.75; H, 5.16; N, 8.37; S, 12.16.

EXAMPLE 1777-(Phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicAcid

A suspension of sodium cephalothin (3.36 g.) in anhydrous alcohol-freechloroform (20 ml.) is silylated by the addition oftrimethylchlorosilane (2.2 ml.). After stirring for 30 minutes,monosilyltrifluoroacetamide (5.0 ml.) and phenylacetyl chloride (4.0ml.) are added and the mixture is then heated to 45° C. for two daysunder a condenser fitted with a drying tube. The volatiles areevaporated to afford a residue which is dissolved in 100 ml. of ethylacetate and washed three times with water. The ethyl acetate layer isdried over magnesium sulfate, filtered and the solution evaporated invacuo to a residue. The residue is triturated with chloroform, anyinsolubles are removed by filtration and the product precipitated fromthe filtrate with hexane. This procedure is followed two more times. The7-(phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicacid is obtained in a solvent-free form by freeze drying from a solutionin benzene.

IR: (CHCl₃) 1780μ 1720μ ##STR100##

TLC: 1 major spot, R_(f) =0.69 (EtOAC:62 , C₅ H₅ N:21, HOAC:6, H₂ O:11)on silica gel.

EXAMPLE 1787-(Di-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic Acid

A suspension of sodium cephalothin (1.18 g.) in anhydrous alcohol-freechloroform (10 ml.) is silylated by the addition oftrimethylchlorosilane (1.1 ml.). After stirring for 30 minutes,monosilyltrifluoroacetamide (2.5 ml.) and 2-thienylacetyl chloride (2.0ml.) are added to the suspension which is then heated to 45° C. Themixture is allowed to remain at this temperature for two days and thenevaporated in vacuo to a residue which is dissolved in ethyl acetate (50ml.) and washed three times with water. The ethyl acetate layer is driedover magnesium sulfate, filtered and the filtrate evaporated in vacuo toobtained a residue. The residue is dissolved in chloroform andprecipitated with hexane three times, each time discarding thesupernatent liquid. The7-(di-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic acidis obtained in a solvent-free form by freeze-drying from a solution inbenzene.

NMR: (Solvent-CDCl₃)-Consistent with structure; ##STR101##

TLC: 1 major spot, R_(f) =0.67 (EtOAC:62, C₅ H₅ N:21, HOAC:6, H₂ O:11)on silica gel.

EXAMPLE 1797-(2-Thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic Acid

7-(Phenylacetyl-2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylicacid (250 mg.) is dissolved in tetrahydrofuran (10 ml.) and water(10ml.). The pH of the solution is adjusted to 9 and the mixture isallowed to stand for one hour. After this the solution is extracted withethyl acetate and the extracts are washed with a disodium hydrogenphosphate solution. After drying the solvent is evaporated to afford amixture of the7-(2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic acid andthe starting material. The product is separated from the startingmaterial by chromatography to afford substantially pure7-(2-thienylacetyl)amino-3-acetoxymethyl-3-cephem-4-carboxylic acid. Theratio of these two products is 7:3.

EXAMPLE 180

By following substantially the procedure described in Example 170 StepA, and by substituting for the thienylacetyl chloride described thereinan equimolar quantity of an appropriate acylating agent there isobtained the corresponding diester of7-diacylamino-3-carbamoyloxymethyl-7-methoxy(orhydro)-3-cephem-4-carboxylic acid which upon removal of the N-protectinggroup by following the procedure of Example 170, Step B, and subsequenttreatment by the procedure of Step C affords the desired7-acylamino-3-carbamoyloxymethyl-7-methoxy(orhydro)-3-cephem-4-carboxylic acid.

The following equation, taken together with Table I, illustrates thestarting materials, intermediates and novel final products which can beprepared by this novel process. It should be noted that in the followingequation and table that those compounds having a 7-hydro substituentinstead of the 7-methoxy substituent would undergo the same reaction toafford those compounds of Table I having a 7-hydro substituent in placeof the 7-methoxy substituent. ##STR102##

                  TABLE I                                                         ______________________________________                                        R.sup.1                                                                       ______________________________________                                         ##STR103##                                                                    ##STR104##                                                                    ##STR105##                                                                    ##STR106##                                                                   CH.sub.3 CHCHCH.sub.2                                                         C.sub.2 H.sub.5 SCH.sub.2                                                     n-C.sub.4 H.sub.9 SCH.sub.2                                                   CH.sub.2CHSCH.sub.2                                                            ##STR107##                                                                    ##STR108##                                                                    ##STR109##                                                                   CH.sub.3 CH.sub.2 CHCHCH.sub.2                                                n-C.sub.5 H.sub.9                                                             n-C.sub.6 H.sub.11                                                            C.sub.2 H.sub.5                                                                ##STR110##                                                                    ##STR111##                                                                    ##STR112##                                                                    ##STR113##                                                                    ##STR114##                                                                    ##STR115##                                                                    ##STR116##                                                                    ##STR117##                                                                    ##STR118##                                                                    ##STR119##                                                                   CNCH.sub.2                                                                     ##STR120##                                                                    ##STR121##                                                                    ##STR122##                                                               

EXAMPLE 181 7-Amino-7-Allyl-Cephalosporanic Acid Benzhydryl Ester7-Amino-7-Benzyl-Cephalosporanic Acid Benzhydryl Ester

The benzhydryl ester of 7-(p-nitrobenzylideneamino)-cephalosporanic acidis reacted with allyl chloride, following the procedure of Example 166,to form benzhydryl 7-(p-nitrobenzylideneamino)-7-allyl-cephalosporanate.This is then regenerated to the benzhydryl7-amino-7-allyl-cephalosporanate using aniline hydrochloride.

The benzhydryl 7-amino-7-benzyl-cephalosporanate is prepared in the samemanner using benzyl chloride.

EXAMPLE 182 7-Amino-7-Carboxy-Cephalosporanic Acid Benzhydryl Ester7-Amino-7-Dithiocarboxy-Cephalosporanic Acid Benzhydryl Ester

A solution of 0.5 g. of benzhydryl7-(p-nitrobenzylideneamino)-cephalosporanate in 5 ml. of tetrahydrofuranis prepared under a nitrogen atmosphere. Carbon dioxide gas is bubbledthrough the solution until the color disappears. Benzene, 50 ml., isadded and the solution washed with aqueous pH 2 phosphate buffer. Thebenzene solution is dried with MgSO₄, filtered, and evaporated to affordthe product, benzhydryl7-(p-nitrobenzylideneamino)-7-carboxy-cephalosporanate.

Using carbon disulfide gas or dry ice (solid CO₂) in the above reaction,the compounds7-(p-nitrobenzylideneamino)-7-dithiocarboxy-cephalosporanate or7-(p-nitrobenzylideneamino)-7-carboxy-cephalosporanate are prepared,respectively.

The amino moiety is regenerated using aniline hydrochloride, therebyyielding the compounds 7-amino-7-carboxy-cephalosporanic acid benzhydrylester or 7-amino-7-dithiocarboxy-cephalosporanic acid benzhydryl ester.

EXAMPLE 183 7-Amino-7-Nitro-Cephalosporanic Acid Benzhydryl Ester

Benzhydryl 7-(p-nitrobenzylideneamino)-cephalosporanate, 571 mg., isdissolved in 10 ml. dry acetonitrile containing 4 mg. acetone cyanhydrinnitrate. The latter has the formula: ##STR123## With vigorous stirringunder nitrogen, a solution of diisopropylethylamine in 12 ml.acetonitrile is added over a five hour period. The reaction mixture isstirred overnight and the solvent removed in vacuo. The residue is takenup in 50 ml. benzene and washed successively with water, 1 M aqueous pH2 phosphate buffer, water and bicarbonate. After drying, filtering andremoving the solvent, the residue is a crude compound suitable forfurther reaction without additional purification. It may be purified ifdesired by chromatography on silica gel, eluting with 25:1chloroform-ethyl acetate. It is identified as7-(p-nitrobenzylideneamino)-7-nitro-cephalosporanic acid benzhydrylester.

The compounds 7-(p-nitrobenzylideneamino)-7-nitroso-cephalosporanic acidbenzhydryl ester;7-(p-nitrobenzylideneamino)-7-carbamoyl-cephalosporanic acid benzhydrylester; 7-(p-nitrobenzylideneamino)-7-carboethoxy-cephalosporanic acidbenzhydryl ester; 7-(p-nitrobenzylideneamino)-7-sulfo-cephalosporanicacid benzhydryl ester;7-(p-nitrobenzylideneamino)-7-sulfamoyl-cephalosporanic acid benzhydrylester; 7-(p-nitrobenzylideneamino)-7-methylsulfo-cephalosporanic acidbenzhydryl ester; or7-(p-nitrobenzylideneamino)-7-phospho-cephalosporanic acid benzhydrylester can be prepared using the reagents nitrosyl chloride, carbamoylchloride, ethylchloroformate, sulfamoyl chloride, methanesulfonylchloride, or phosphorous oxychloride, respectively.

The above imino derivatives can all be regenerated to the aminofunctionality using either aniline hydrochloride or2,4-dinitrophenylhydrazine, as described infra. The products therebyobtained are 7-amino-7-nitro-cephalosporanic acid,7-amino-7-nitrosocephalosporanic acid,7-amino-7-carbamoyl-cephalosporanic acid,7-amino-7-carboethoxy-cephalosporanic acid,7-amino-7-sulfo-cephalosporanic acid, 7-amino- b7-sulfamoylcephalosporanic acid, 7-amino-7-methylsulfo-cephalosporanicacid and 7-amino-7-phospho-cephalosporanic acid, respectively. In allcases, the benzhydryl ester is the ester made.

EXAMPLE 184 Benzhydryl 7-aminocephalosporanate-S-oxide

0.500 g. of benzhydryl 7-aminocephalosporanate is dissolved in 10 ml. ofCH₂ Cl₂ cooled to 0° C. and treated with m-chloroperbenzoic acid (0.172g.) for 1 hour, during which the reaction mixture is allowed to come toroom temperature.

The reaction mixture is diluted with CH₂ Cl₂ and washed with 5% NaHCO₃solution three times and then with water, dried and evaporated to givethe crude product. This is stirred with 10 ml. of ether for 1 hour. Theprecipitate is filtered and washed with ether to give the title product.

In like manner other 7-aminodecephalosporanic acid esters describedherein can be converted to the S-oxides. These oxides can be used asintermediates in the preparation of the sulfoxides set forth in formulaI above. Alternatively, the various intermediates and final productsdescribed herein can be converted to the corresponding S-oxidesfollowing the procedure described above. The various S-oxide derivativescan be converted to the corresponding cephem compounds in accordancewith the process of the example which follows.

EXAMPLE 185

Benzhydryl3-methoxymethyl-7-methoxy-7-(2-thienylacetamido))cephalosporanate-S-oxideprepared by the oxidation of the corresponding cephalosporanate compoundwith m-chloroperbenzoic acid (1.0 g.) is dissolved in 10 ml. ofacetonitrile and 10 ml. of dimethylformamide. An equivalent of stannouschloride and 1/2 equivalent of acetyl chloride is added at 0° C. Themixture is stirred at 0° C. for 1 hour and then at room temperature for1 hour. The solvent is removed in vacuo and water is added. The mixtureis extracted with ethylacetate and the extract washed with 0.1 N HCl,saturated sodium bicarbonate and water. After drying the solvent isremoved in vacuo to afford benzhydryl3-methoxymethyl-7-methoxy-7-(2-thienylacetamido)cephalosporanate.

The new cephalosporins of formula I are valuable antibiotics activeagainst various gram-positive and gram-negative bacteria. Although, ingeneral, their biological spectrums are similar to those of the knowncephalosporins, these new cephalosporins possess some new and unexpectedproperties. Thus, in general, they are active against manymicroorganisms which are resistant to the known cephalosporins such ascephaloridine and cephalothin and are resistant to the β-lactamaseproduced by cephalosporin resistant clinical isolates of pathogens suchas E. coli and A. cloacae. Also, they are generally more active againststrains of Proteus such as mirabilis, and are active against strains ofProteus morganii which are resistant to the unsubstitutedcephalosporins. They are useful in separating microorganisms inremaining susceptible microorganisms from pharmaceutical, medical anddental equipment and as bactericides in industrial applications, forexample in water-based paints and in the white water of paper mills toinhibit the growth of harmful bacteria.

Thus, the 7-methoxycephalosporins produced in accordance with theprocesses of this invention are generally more active than the7-(D-5'-amino-5'-carboxyvaleramido)-7-methoxycephalosporins againstvarious gram-negative organisms and possess increased activity againstgram-positive organisms. For example, these 7-methoxycephalosporins areactive against gram-positive pathogens such as Staphylococcus aureus atMinimum Inhibitory Concentrations (MIC) as low as about 1.5 mcg./ml.,Streptococcus pyogenes at MIC of about 0.7 mcg./ml., and Diplococcuspneumoniae at MIC of about 0.7 mcg./ml.; and against gram-negativeorganisms such as Aerobacter aerogenes at MIC of about 3 mcg./ml.,Proteus vulgaris at MIC of about 1.5 mcg./ml. and Proteus morganii atabout 6 mcg./ml. Thus, activities of specific products of the foregoingexamples that might be mentioned are:3-carbamoyloxymethyl-7-methoxy-7-phenylacetamido-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 0.78 mcg./ml. and P. morganii MIC 12.5 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-(2-furylacetamido)-3-cephem-4-carboxylicacid, S. aureus MIC 6.25 mcg./ml. and P. vulgaris MIC 1.56 mcg./ml.;3-carbamoyloxymethyl-7-methoxy-7-thiophenoxyacetamido-3-cephem-4-carboxylicacid, S. pyrogenes MIC 0.78 mcg./ml. and D. pneumoniae MIC 0.78mcg./ml.;3-acetoxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid, S. pyogenes MIC 1.56 mcg./ml. and P. vulgaris MIC 0.78 mcg./ml;and3-pyridiummethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid Serratia MIC 25 mcg./ml. and S. aureus MIC 156 mcg./ml.

The products of this invention may be used alone or in combination asthe active ingredient in any one of a variety of pharmaceuticalpreparations. These antibiotics and their corresponding salts may beemployed in capsule form or as tablets, powders or liquid solutions oras suspensions or elixirs. They may be administered orally,intravenously or intramuscularly. Suitable carriers which may be used inthe composition include, for example, mannitol, sucrose, glucose orsterile liquids such as water, saline, glycols and oils of a petroleum,animal, vegetable or synthetic origin as, for example, peanut oil,mineral oil or sesame oil. Also, in addition to a carrier, the instantcompositions may include other ingredients such as stabilizers, binders,antioxidants, preservatives, lubricators, suspending agents, viscosityagents or flavoring agents and the like. In addition, there may also beincluded in the composition other active ingredients to provide abroader spectrum of antibiotic activity.

The dosage to be administered depends to a large extent upon thecondition of the subject being treated and the weight of the host, theparenteral route being preferred for generalized infections and the oralroute for intestinal infections. In general, a daily dosage consists offrom about 15 to about 600 mg. of active ingredient per kg. of bodyweight of the subject in one or more applications per day. A preferreddaily dosage lies in the range of from about 80 to 120 mg. of activeingredient per kg. of body weight. The preferred daily dosage for thecompound sodium3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)decephalosporanateis in the range of from about 80 to 120 mg. of active ingredient per kg.of body weight.

The instant compositions may be administered in several unit dosageforms as, for example, in solid or liquid orally ingestible dosage form.The compositions per unit dosage, whether liquid or solid, willgenerally contain from about 15 mg. to about 1500 mg. by weight of theactive ingredient based upon the total weight of the composition;however, in general, it is preferable to employ a dosage amount in therange of from about 250 mg. to 1000 mg. In parenteral administration theunit dosage is usually the pure compound in a slightly acidified sterilewater solution or in the form of a soluble powder intended for solution.Typical formulations of specific products are described below.

One such unit dosage form consists in mixing 120 mg. of3-carbamoyloxymethyl-7-methoxy-7-(D-α-amino-phenylacetamido)decephalosporanicacid sodium salt with 20 mg. of lactose and 5 mg. of magnesium stearateand placing the 145 mg. mixture into a No. 3 gelatin capsule. Similarly,by employing more of the active ingredient and less lactose, otherdosage forms can be put up in No. 3 gelatin capsules and should it benecessary to mix more than 145 mg. of ingredients together, largercapsules such as compressed tablets and pills can also be prepared. Thefollowing examples are illustrative:

    ______________________________________                                        Tablet Containing 125 mg. of 7-Methoxy-7-(D-α-carboxy-                  Phenylacetamido)cephalosporanic Acid                                                           Per Tablet                                                   ______________________________________                                        7-Methoxy-7-(D-α-Amino-                                                 Phenylacetamido)cephalo-                                                      sporanic Acid      125.       mg.                                             Cornstarch, U.S.P. 6.         mg.                                             Dicalcium Phosphate                                                                              192.       mg.                                             Lactose, U.S.P.    190.       mg.                                             ______________________________________                                    

The active ingredient is blended with the dicalcium phosphate, lactoseand about half of the cornstarch. The mixture is then granulated with a15% cornstarch paste (6 mg.) and rough-screened. It is dried at 45° C.and screened again through No. 16 screens. The balance of the cornstarchand the magnesium stearate is added and the mixture is compressed intotablets, approximately 0.5 inch in diameter each weighing 800 mg.

    ______________________________________                                        Parenteral Solution Containing 500 mg. of 3-Carbamoyl-                        oxymethyl-7-Methoxy-7-(2-Thienylacetamido)decephalosporanate                  Ampoule:                                                                      ______________________________________                                        Sodium 3-Carbamoyloxymethyl-7-Methoxy-                                        7-(2-Thienylacetamido)de-                                                     cephalosporanate         500    mg.                                           Diluent: Sterile Water for Injection                                                                   2      cc.                                           ______________________________________                                    

By substituting an equivalent amount of7-methoxy-7-(α-carboxy-phenylacetamido)cephalosporanic acid for the 500mg. of sodium salt of3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)decephalosporanaterecited in the foregoing example there is also obtained a formulationsuitable for parenteral administration.

    ______________________________________                                        Opthalmic Solution Containing 100 mg. of Sodium 3-Carbam-                     oyloxymethyl-7-Methoxy-7-(2-Thienylacetamido)                                 decephalosporanate                                                            ______________________________________                                        Sodium 3-Carbamoyloxy-7-Methoxy-7-                                            (2-Thienylacetamido)decephalo-                                                sporanate                100    mg.                                           Hydroxypropylmethyl Cellulose                                                                          5      mg.                                           Sterile Water            to 1   ml.                                           ______________________________________                                        Otic Solution Containing 100 mg. of Sodium 3-Carbamoyloxy-                    methyl-7-Methoxy-7-(2-Thienylacetamido)decephalosporanate                     ______________________________________                                        Sodium 3-Carbamoyloxy-7-Methoxy-7-                                            (2-Thienylacetamido)decephalo-                                                sporanate                100    mg.                                           Benzalkonium Chloride    0.1    mg.                                           Sterile Water            to 1   ml.                                           ______________________________________                                        Topical Ointment Containing 100 mg. of Sodium 3-Carbamoyloxy-                 methyl-7-Methoxy-7-(2-Thienylacetamido)decephalosporanate                     ______________________________________                                        Sodium 3-Carbamoyloxy-7-Methoxy-7-                                            (2-Thienylacetamido)decephalo-                                                sporanate                100    mg.                                           Polyethylene Glycol 4000 U.S.P.                                                                        400    mg.                                           Polyethylene Glycol 400 U.S.P.                                                                         1.0    gram                                          ______________________________________                                    

The active ingredient in the above formulations may be administeredalone or in combination with other biologically active ingredients as,for example, with other antibacterial agents such as lincomycin, apenicillin, streptomycin, novobiocin, gentamicin, neomycin, colistin andkanamycin, or with other therapeutic agents such as probenecid.

The new cephalosporins of this invention can be used in the form of thefree acid, as salts such as alkali metal, alkaline earth metal orammonium salts, for example, sodium, potassium, calcium,triethylammonium, procaine, and the like, as esters, particularly labileesters such as acetoxymethyl or pivaloyloxy and the like, or as amides.

The7-(D-5'-amino-5'-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid and the derivatives thereof having various substituents at the3-position of the general formula --CH₂ A as defined above are disclosedand claimed in the following United States applications of Edward O.Stapley and Justo M. Mata:

Ser. No. 19,496 filed Mar. 13, 1970

Ser. No. 51,319 filed June 30, 1970

Ser. No. 96,594 filed Dec. 9, 1970 and

Ser. No. 115,779 filed Feb. 16, 1971.

These products are therefore not within the scope of this invention.

What is claimed is:
 1. An antibacterial composition comprising anantibacterially effective amount of a3-A-7-methoxy-7-acylamido-3-cephem-4-carboxylic acid wherein A ismethyl, hydroxymethyl, chloromethyl, bromomethyl, or fluoromethyl,mercaptomethyl, methoxymethyl, n-propoxymethyl, methylthiomethyl,acetoxymethyl, propionyloxymethyl, benzoyloxymethyl,(p-chlorobenzoyl)oxymethyl, (p-methylbenzoyl)oxymethyl,pivaloyloxymethyl, (1-adamantyl)carboxymethyl, butanoyloxymethyl,carbamoyloxymethyl, (N-methylcarbamoyl)oxymethyl,(N-ethylcarbamoyl)oxymethyl, [N-(2-chloroethyl)carbamoyl]oxymethyl,(N-phenylcarbamoyl)oxymethyl, N-p-sulfophenylcarbamoyl)oxymethyl,p-carboxymethylphenylcarbamoyloxymethyl, methoxycarbonyloxymethyl,isobutanoyloxymethyl, cyclobutylcarbonyloxymethyl, carbamoylthiomethyl,(ethoxythiocarbonyl)thiomethyl, (n-propoxythiocarbonyl)thiomethyl,(cyclopentanoxythiocarbonyl)thiomethyl,N,N-diethylthiocarbamoylthiomethyl,N-methylpiperazinium-1-thiocarbonylthiomethyl,N,N-dimethylpiperzinium-1-thiocarbonylthiomethyl, 2-furoylthiomethyl,isothiouroniummethyl, (5-methyl-1,3,4-thiadiazol-2-yl)-thiomethyl,p-tolylsulfonylthiomethyl, mesyloxymethyl,1-methyl-1,2,3,4-tetrazolyl-5-thiomethyl, tosyloxymethyl,sulfamoyloxymethyl, 1-naphthoyloxymethyl, 2-furylacetoxymethyl,cinnamoyloxymethyl, p-hydroxycinnamoyloxymethyl,p-sulfocinnamoyloxymethyl and 1R:2S-epoxypropylphosphonyloxymethyl, orpyridinium methyl; and acylamido is ##STR124## wherein n is 0-4, Z isoxygen or sulfur, and R" is benzyl, p-hydroxybenzyl,4-amino-4-carboxybutyl, methyl, cyanomethyl, 2-pentenyl, n-amyl,n-heptyl, ethyl, 3- or 4-nitrobenzyl, phenethyl, β,β-diphenylethyl,methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl,2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, 3,5-dimethyl-4-isoxazolyl,3-butyl-5-methyl-4-isoxazolyl, 5-methyl-3-phenyl-4-isoxazolyl,3-(2-chlorophenyl)-5-methyl-4-isoxazolyl,3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl, D-4-amino-4-carboxybutyl,D-4-N-benzoylamino-4-carboxy-n-butyl, p-aminobenzyl, o-aminobenzyl,m-aminobenzyl, (3-pyridyl)-methyl, 2-ethoxy-1-naphthyl,3-carboxy-2-quinoxalinyl,3-(2,6-dichlorophenyl)-5-(2-furyl)-4-isoxazolyl, 3-phenyl-4-isoxazolyl,5-methyl-3-(4-guanidinophenyl)-4-isoxazolyl, 4-guanidinomethylphenyl,4-quanidinomethylbenzyl, 4-guanidinobenzyl, 4-quanidinophenyl,2,6-dimethoxy-4-guanidinophenyl, o-sulfobenzyl, p-carboxymethylbenzyl,p-carbamoylmethylbenzyl, m-fluorobenzyl, m-bromobenzyl, p-chlorobenzyl,p-methoxybenzyl, 1-naphthylmethyl, 3-isothiazolylmethyl,4-isothiazolylmethyl, 5-isothiazolylmethyl, 4-pyridylmethyl,5-isoxazolylmethyl, 4-methoxy-5-isoxazolylmethyl,4-methyl-5-isoxazolylmethyl, 1-imidazolylmethyl, 2-benzofuranylmethyl,2-indolylmethyl, 2-phenylvinyl, 2-phenylethynyl,2-(5-nitrofuranyl)vinyl, phenyl, o-methoxyphenyl, o-chlorophenyl,o-phenylphenyl, p-aminomethylbenzyl, 1-(5-cyanotriazolyl)methyl),difluoromethyl, dichloromethyl, dibromomethyl,1-(3-methylimidazolyl)methyl, 2- or 3-(5-carboxymethylthienyl)methyl, 2-or 3-(4-carbamoylthienyl)methyl, 2- or 3-(5-methylthienyl)methyl, 2- or3-(5-methoxythienyl)methyl, 2- or 3-(4-chlorothienyl)methyl, 2- or3-(sulfothienyl)methyl, 2-or 3-(5-carboxythienyl)methyl,3-(1,2,5-thiadiazolyl)methyl, 3-(4-methoxy-1,2,5-thiadiazolyl)methyl,2-furylmethyl, 2-(5-nitrofuryl)methyl, 3-furylmethyl, 2-thienylmethyl,3-thienylmethyl, and tetrazolylmethyl; and the pharmaceuticallyacceptable salts thereof, or the carboxylic acid protecting derivativesthereof wherein the derivative group is methyl, t-butyl, trichloroethyl,allyl, propargyl, benzyl, diphenylmethyl, o-nitrobenzyl,3,5-dinitrobenzyl, p-methoxybenzyl, acetoxymethyl, pivaloyloxymethyl,phenacyl, trichloroethoxy carbonyl, trimethylsilyl or tributyltin in apharmaceutically acceptable carrier vehicle.
 2. The composition of claim1 wherein the cephem-4-carboxylic acid is3-carbamoyloxymethyl-7-methoxy-7-(2-thienylacetamido)-3-cephem-4-carboxylicacid or a salt thereof.
 3. The composition of claim 2 wherein the saltis the sodium salt.